2021 Volume 44 Issue 11 Pages 1707-1716
Mupirocin nanoparticle-loaded hydrogel (MLH) was successfully developed. This study focused on the safety of cell lines and the biocompatibility of MLH for wound healing in rat models. MLH was assessed by an analysis of cytotoxicity and the secretion of inflammatory cytokines in cell lines. The cytocompatibility of MLH was compared with mupirocin ointment on full-thickness burn wounds in rats. The results indicated that MLH and blank hydrogel had no toxicity to human epidermal keratinocytes and human fibroblast cells. MLH inhibited lipopolysaccharide (LPS) activity in macrophage-like cells resulting in low nitric oxide production and reduced inflammatory cytokine production (interleukin (IL)-1β) compared with a positive control (LPS only). In burn wounds, MLH and hydrogel healed the wound better than the other groups determined by wound contraction, reduced secretion, and the generation of new blood vessels, as well as promotion of hair follicle cells. Better granulation tissue proliferation, less necrosis, and a lower degree of inflammation were found in the MLH and blank hydrogel than in the mupirocin ointment. The hydrogel group reduced the macrophages (CD68) on day 14 at the edge of the wound. On day 28, T cells (CD3), B cells (CD20), and CD68+ cells were concentrated in the deeper subcutaneous tissue. Additionally, the transforming growth factor β1 (TGF-β1) concentration and matrix prometalloproteinase-2/tissue inhibitor of metalloproteinases-2 ratio in the MLH and hydrogel groups were less than those in the other groups. The MLH formulation was safe and effective in burn wound healing. Therefore, MLH formulations are promising candidates for further evaluation in clinical trials.
The skin function as a barrier for protection from surrounding. The skin and skin morphology can change after surgery, trauma, ischemia, burns, or other wounds that affect skin dysfunctions.1) A burn wound can be induced by heat, chemical, and electrical in which time, temperature, concentration and electrical power determine the extent of the damage.2) A burn wound can be defined as superficial partial, deep partial and full thickness.2) After the time of injury, Gram-positive bacteria, such as Staphylococcus aureus and Corynebacterium and Streptococcus species, colonize at the surface of the wound.3) Mupirocin is often used to treat topical infections caused by S. aureus and S. epidermidis, including Methicillin-resistant Staphylococcus aureus (MRSA). Mupirocin 2% ointment is commercially available in the market.4) Nevertheless, the ointment base is present the disadvantages as greasiness, stickiness, and viscosity-temperature variations that are undesirable for patients.5) Researchers have attempted to develop mupirocin in other forms.6–8) For example, liposomal mupirocin was shown to have improved therapeutic effects and reduced toxicity compared with mupirocin only.6) However, some limitations still exist. For instance, during sterilization liposomes are sensitive to high temperatures. As a consequence, liposomes release the drug when the temperature rises above room temperature. Furthermore, lipids or other compositions of liposomes, or both can become toxic.9)
Hydrogels are polymer networks, which are produced by chemical or physical interactions among the polymer chains that can retain huge high water in their structures.10,11) Hydrogels are of particular interest in wound healing because they can absorb and retain wound exudates. Therefore, hydrogels promote fibroblast proliferation and keratinocyte migration that are important for epithelialization in wound healing.12,13) An amorphous hydrogel is preferred for cavity wounds and finds application mainly in the treatment of superficial burns. An application of hydrogels to the wound is the way to cool the wound to relieve damage and reduce pain, provided that clean and safe water are not obtainable for first-aid in the case of burn wounds.14) Furthermore, hydrogels have excellent hydrophilic and biocompatibility properties.
In our previous communication, a mupirocin nanoparticle-loaded hydrogel formulation (MLH) was successfully developed.15) Mupirocin can release through various mechanisms, including diffusion, swelling, and chemically controlled methods, in hydrogel systems.16) MLH drug release from nanoparticles and hydrogel systems can permeate the cell wall and bind to bacterial isoleucyl-tRNA synthetase.4) Therefore, the safety and efficacy of MLH in experimental full-thickness burn wounds compared with a conventional ointment were evaluated. The histology, biomarkers for wound healing, and immunohistochemical profile were recorded in this study.
Cell viability was evaluated following treatment with MLH, mupirocin ointment, mupirocin, and blank hydrogel on human epidermal keratinocyte (HaCaT) cell (Cell Lines Service GmbH, Eppelheim, Germany) and human fibroblast (BJ) cell (ATC C, Manassas, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM) was the culture mediums for the HaCaT and BJ cells, respectively. Fetal bovine serum (FBS, 10%) and 100 U/mL of penicillin–streptomycin as antibiotics were added to the culture mediums at 37 °C in an atmosphere of 5% CO2. After the cells were grown in a full flask, they were harvested by trypsinization in 4 mL of Versene-trypsin buffer. Before cells were seeded into a 96-well plate, these cells were centrifuged at 2000 rpm at 4 °C for 5 min and approached to 106 cells mL−1. The methyl thiazol tetrazolium (MTT) assay was employed to examine the toxicity.17) The control was the culture medium. The experiment was started by dissolving 1 mg of mupirocin or 1 mg mupirocin equivalent in MLH or mupirocin ointment (mupirocin in a water-soluble ointment base) in 2 mL of culture medium to produce a drug concentration of 500 µg mL−1. An amount of 100 µL was loaded into each well of the culture medium that had been seeded. Incubation was then carried out for 24 h. Fresh medium (100 µL) was used to replace the old culture medium and then 50 µL of MTT solution was added. Incubation was for 4 h at 37 °C in 5% CO2 incubator. The culture media were removed and dimethyl sulfoxide (100 µL) was placed into the well. A microtiter plate reader (Biohit 830, Helsinki, Finland) was used to evaluate the absorbance (abs) at 570 nm. The following equation was used to determine cell viability:
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Assessments of inflammatory cytokine production (interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α)) and NO release from RAW264.7 cells were performed after Escherichia coli lipopolysaccharide (LPS) stimulation. The RAW264.7 cells were seeded into a 96-well plate to obtain a cell concentration of 106 cells/well and incubated at 37 °C under 5% CO2 for 24 h. The old medium was then replaced with fresh medium. The cells were stimulated with LPS at a concentration of 10 µg mL−1 in the presence or absence of MLH or mupirocin ointment for 18 h. An amount of 50 µL of the supernatant was collected for an assay of NO. The supernatant was mixed with Griess reagent (100 µL) and incubated for 10 min at room temperature. A microplate reader was used to evaluate NO using the absorbance at a wavelength of 540 nm. Whereas, the inflammatory cytokine level was measured using UV–vis absorbance at 450 nm. The quantitative determination of IL-1β and TNF-α was determined using mouse IL-1β and mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, U.S.A.).
In Vitro Scratch AssayThe scratch assay was used to assess the wound healing capabilities by observing BJ cell migration. The BJ cells were seeded in 6 well plates to obtain a cell concentration of 106 cells/well. After a monolayer was formed, the cells were scratched with a p1000 pipet tip and then washed using 1 × phosphate-buffered saline. The old medium was replaced with 1 mg mupirocin or 1 mg mupirocin equivalent in MLH or mupirocin ointment in 2 mL of culture medium to make a drug concentration of 500 µg mL−1. The control was cells without any treatment. Images from microscope were taken at 0, 24, 48, and 72 h and new medium containing mupirocin was replaced after an image was taken at each time point to keep the medium content constant. NIH Image of the ImageJ program (1.42q/Java 1.8.0.1121) was then used to quantitatively measure all scratch wound closures. The results were reported as a percentage of the distance covered by the migrating cells.18)
Collagen ProductionThe production of collagen after treatment with MLH, mupirocin, ointment, mupirocin, and blank hydrogel was investigated using the BJ cells. The collagen production test used cultured cells as explained by the scratch assay. The cells were seeded at 106 cells/well in a 96-well plate. Type I collagen production was examined by the Sircol™ collagen assay kit (Biocolor Ltd., County Antrim, U.K.). Precisely, 1 mg mupirocin or 1 mg mupirocin equivalent in MLH or mupirocin ointment in 2 mL of culture medium was used to make a drug concentration of 500 µg mL−1 which was added to the cells and incubated for 24 h. The control was the culture medium. The dye reagent (300 µL) was then added to supernatant (100 µL) at room temperature for 10 min. All samples were centrifuged for 10 min at 15000 × g and then we removed the supernatant. An alkali indicator reagent (500 µL) was used to re-dissolve the pellets. Measurements were done by a microplate reader at 540 nm. A standard curve of collagen concentration was used to calculate the collagen production.
In Vivo Animal StudiesAnimal PreparationAll protocols were followed according to the guideline approved by the Animal Ethics Committee, Prince of Songkla University (Ref. 61/2019). Twenty-four male Sprague Dawley rats (280–330 g, 6 weeks old) were obtained from the Nomura Siam International Co., Ltd., Bangkok, Thailand. The rats were corralled at 25 ± 2 °C, under 12 h : 12 h light–dark cycle, and given ad libitum access to standard rodent diets and water. The rats were acclimatized in these conditions for seven days before use. The rats were anesthetized by intraperitoneal injection with 5 mg/kg of Zoletil 100® containing 250 mg of tiletamine and 250 mg of zolazepam (Virbac, Carros, France). The dorsal hair of the rats was shaved with an electric shaver. The area of the burn inflictions was 6 × 4 cm. The rat dorsum was marked by lines that divided the dorsum into four equal areas. Each quadrant accommodated a single burn wound The skin was disinfected with povidone-iodine and then left to dry at room temperature for 3 min before burn infliction.
Burn Wound CreationA square stainless-steel rod of 1 cm2 area was used for infliction of the burns. The temperature of the square stainless-steel rod was monitored by a thermocouple. The rod was immersed in a gas cartridge burner and heated to 150 ± 5 °C and then placed onto the rat skin to create a full-thickness burn for 10 s.
Sample TreatmentsTwenty-four male rats were randomly assigned into four groups: normal saline solution (NSS), mupirocin ointment, blank hydrogel, and MLH. The animals were topically treated with 50 mg blank hydrogels to the upper left wound and 50 mg of 2% (w/w) MLH formulation were applied to the upper right wound. A commercially available product of mupirocin as 50 mg of 2% (w/w) mupirocin ointment was applied to the lower left wound and NSS was applied to the lower right wound. All treatments were equivalent to 1 mg of mupirocin. All wounds were not covered with tape and cleaned once every three days using sterile NSS. After cleaning, the sample test treatments were reapplied regularly to the burn wounds each day. On days 0, 7, 14, and 28, tissue samples were collected from all wounds of the six rats from each group for inflammatory mediators and histological evaluation. Six rats from each group were kept for 28 d after treatment to observe wound healing.
Wound Size Measurement and Morphological EvaluationWound size was measured by taking daily photographs for a total of 28 d. A rectangular reference with an area of 1 cm2 was placed parallel to permit correction for the distance between the camera and the rats. The wound area was calculated by photography combined with ImageJ software and expressed as a percentage of wound contraction compared to the original area using the following equation:
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where A0 is the initial wound area and At is the wound area at the time of observation. The clinical criteria for the morphological evaluation of wound healing were scored according to secretion type, wound secretion, wound color, and scar formation.
Histologic AnalysisRats from a selected group were euthanized using a high dose of pentobarbital sodium. Their skin tissue was then collected from the wounds on days 0, 7, 14, and 28. The skin tissue was cut and fixed in 10% neutral buffered formalin. From each paraffin embedded specimen, thin sections (3 µm) were prepared on each slide and stained with a hematoxylin–eosin (H&E) staining kit (Sigma-Aldrich, St. Louis, MO, U.S.A.) according to the standard H&E staining protocol. The wound healing effects were examined by histological study under a light microscope equipped with a digital camera (BX61, Olympus, Tokyo, Japan) at 20× and 40×. The histological parameters for assessment of healing were the features of the post-burn full-thickness wounds on days 0, 7, 14, and 28 in the treatment of NSS, blank hydrogel, MLH, and mupirocin ointment. On day 14, the blood vessel density was employed to interpret quantification of the vascularization. At the beginning, we defined the cross-sectional area of tissue samples in the image. Then, the number of blood vessels was computed. The area was divided by the number of blood vessel density, after that the average blood vessel density per group was reported. Moreover, at day 21 after the burn, we assessed each wound histologically for hair follicles.
Immunohistochemical AnalysisOn days 0, 7, 14, and 28, samples were collected from each group, fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin, intersected into 5 mm thick perpendicular plane sections, deparaffinized in dimethyl benzene, and rehydrated. The specific antibodies against CD3, CD20, and CD68 were employed to incubate with skin section (LSAB 2 System, DAKO, Germany). Macrophage inflammatory cell infiltration was evaluated by image analysis software (Media Cybernetics, Rockville, MD, U.S.A.) to analyze the quantitative expression levels of the CD3, CD20, and CD68 positive cells.
Biomarkers for Wound Healing QuantificationThe biomarkers for wound healing quantification was performed using total rat matrix prometalloproteinase-2 (MMP-2) (RayBiotech, Norcross, GA, U.S.A.), rat tissue inhibitor of metalloproteinases-2 (TIMP-2) immunoassay ELISA kits (RayBiotech, Norcross, GA, U.S.A.), and rat transforming growth factor β1 (TGF-β1) ELISA kits (Boster Bio-Engineering Limited Company, Wuhan, China) according to the protocols. Three milliliters of extraction buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, and 1% Triton X-100 was employed to homogenize rat skin, which was transferred to 1.5 mL tubes, and then centrifuged at 13000 × g for 15 min at 4 °C. Finally, we stored the supernatant at −80 °C until analyzed.
Statistical AnalysisThe average values and standard deviation (S.D.) of the mean of at least three or six replicates are presented in all experiments. Paired or unpaired Student’s t-tests were employed to determine the statistical differences between groups. The one-way ANOVA was used to analyze the means of the multiple groups identified. A p-value less than 0.05 was considered statistically significant.
The percentages of cell viability of keratinocytes and fibroblast cells after exposure to MLH, blank hydrogel, and mupirocin ointment are shown in Fig. 1a. The keratinocytes and fibroblast cells showed cell viability greater than 80% after exposure of all samples for 24 h. The blank hydrogel had the highest level, followed by MLH and mupirocin ointment. All formulations were non-toxic (shown in Fig. 1a). In the RAW264.7 cells, the percentages of cell viability in the blank hydrogel, and MLH groups were slightly lower than the control group. The blank hydrogel, mupirocin, MLH, and mupirocin ointment did not stimulate NO production. Moreover, the blank hydrogel, MLH, mupirocin, and mupirocin ointment inhibited the production of NO from macrophage cells stimulated by LPS (shown in Fig. 1b). However, inflammatory cytokine production of IL-1β and TNF-α was evaluated on the macrophage cells after treatment with the blank hydrogel, mupirocin, MLH, and mupirocin ointment (shown in Figs. 1c and 1d). Blank hydrogel and MLH slightly stimulated IL-1β and TNF-α production, while mupirocin ointment had a strong potential effect on TNF-α and IL-1β production. The MLH formulation and mupirocin nanoparticles reduced the IL-1β production significantly after exposure to LPS. However, the TNF-α level in RAW264.7 cells did not decrease after MLH was combined with LPS. It is possible that MLH inhibited LPS-induced activation of IL-1β but did not have any effect on TNF-α. Type I collagen production could not be detected in this study. It is possible that collagen production could not be produced from the BJ cells after treatment with mupirocin formulations.
The cell viability was determined by MTT assay. Production of (b) NO and inflammatory cytokines (c) TNF-α and (d) IL-1β from RAW264.7 cells after incubated with hydrogel, mupirocin, MLH, and mupirocin ointment or in combination with 5 µg/mL of LPS. Statistical analysis was done by one-way ANOVA, * p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001. All data represented in mean ± S.D., n = 8.
Wound healing activity was evaluated by examining the migration of the BJ cell line using the in vitro scratch assay at 0, 24, 48, and 72 h (shown in Fig. 2). All formulations showed that closure of the gaps began to occur within 24 to 48 h for the BJ cells. At 72 h of incubation time, the BJ cells of all formulations showed complete closure of the gaps in the blank hydrogel, MLH, and control group, whereas the gap of the mupirocin ointment group was loosely closed.
BJ cells subjected to scratch and treated with (a) hydrogel, (b) MLH, (c) negative control, (d) mupirocin, (e) mupirocin ointment. After incubation, images captured with the magnification power of a 10 × under a light microscope at 0, 24, 48, and 72 h. (mean ± S.D., n = 3.)
Animals were topically treated with MLH, blank hydrogel, mupirocin ointment, and NSS (control group). Reduction in wound size on days 7, 14, and 28 was significantly higher in the treatment groups compared with the control group (shown in Fig. 3). Skin morphology observation and wound size measurements showed that the full-thickness burn wounds completely healed on day 28 for all treatments. Until day 7, the blank hydrogel and MLH showed slightly smaller wound areas than the other treatments, and it was noticeably different from other treatments on day 10. On day 14, a significant difference was observed in wound contraction in the blank hydrogel, MLH, and mupirocin ointment compared to the NSS (p < 0.05). All treatments showed high wound contraction on day 28.
The rats were topically treated with (i) hydrogel on the left upper-sided wound, (ii) 2% (w/w) mupirocin nanoparticle loaded hydrogel (MLH) on the right upper-sided wound, (iii) 2% w/w mupirocin ointment on the left lower-sided wound, and (iv) normal saline solution on the right lower-sided wound (b) Skin photographs of wound contraction in rat dorsal burn on day 0, 3, 5, 7, 10, 14, 21, and 28 after application with NSS, hydrogel, MLH, and mupirocin ointment. Photographs were taken from a representative animal of each group. The representative images were obtained from at least 6 burn. (c) The percentage of wound contraction after treated with NSS, hydrogel, MLH, and mupirocin ointment on full-thickness burns in a rat dorsum on day 3, 5, 7, 10, 14, 21, and 28. (Color figure can be accessed in the online version.)
Morphological assessments of the wound healing criteria were performed after treatment with NSS, blank hydrogel, MLH and mupirocin ointment on full-thickness burns in rat dorsum (Table 1). The data in Table 1 are shown as values of modes of wound secretion, secretion type, wound color and scar formation. From day 3 to day 14 the wound morphologies changed every two days, one week and two weeks. After 14 d the wounds stayed intact in all treatments (days 21 and 28) it meant the wound reached the complete healing stage. On day 3, the secretion was purulent with heavy secretion in all treatments. The scar formation was stiff and it was observed that the wound was similar in MLH and NSS groups, but the wound color of the blank hydrogel and mupirocin ointment groups showed a dark grey. On day 5, there was no different in wound secretion and wound color in each group. Moderate scar formation was observed in the MLH and blank hydrogel groups. However, the NSS and mupirocin ointment groups showed stiff scar formation. On day 7 the hydrogel treatment group was improved in secretion type. Moreover, the blank hydrogel and MLH groups had reduced amounts of wound secretion compared to the NSS and mupirocin ointment groups on day 14. The results showed that there were no secretion after 21 d the wound became bright red with soft scar formation.
| Day | Treatments | Criteria* | |||
|---|---|---|---|---|---|
| Secretion type | Wound secretion | Wound color | Scar formation | ||
| 3 | NSS | 3 | 1 | 3 | 4 |
| Hydrogel | 3 | 1 | 1 | 4 | |
| MLH | 3 | 1 | 3 | 4 | |
| Mupirocin ointment | 3 | 1 | 1 | 4 | |
| 5 | NSS | 1 | 3 | 3 | 2 |
| Hydrogel | 1 | 3 | 3 | 1 | |
| MLH | 1 | 3 | 3 | 1 | |
| Mupirocin ointment | 1 | 3 | 3 | 2 | |
| 7 | NSS | 1 | 3 | 3 | 3 |
| Hydrogel | 2 | 3 | 3 | 2 | |
| MLH | 1 | 3 | 3 | 2 | |
| Mupirocin ointment | 1 | 3 | 3 | 2 | |
| 14 | NSS | 2 | 1 | 4 | 2 |
| Hydrogel | 2 | 4 | 4 | 2 | |
| MLH | 2 | 4 | 4 | 2 | |
| Mupirocin ointment | 2 | 3 | 4 | 2 | |
| 21 | NSS | 4 | 4 | 3 | 3 |
| Hydrogel | 4 | 4 | 3 | 3 | |
| MLH | 4 | 4 | 3 | 3 | |
| Mupirocin ointment | 4 | 4 | 3 | 3 | |
| 28 | NSS | 4 | 4 | 3 | 3 |
| Hydrogel | 4 | 4 | 3 | 3 | |
| MLH | 4 | 4 | 3 | 3 | |
| Mupirocin ointment | 4 | 4 | 3 | 3 | |
* Secretion type (purulent-1, sanguineous-2, serous-3, none-4); wound secretion (heavy-1, moderate-2, low-3, none-4); wound color (dark grey-1, creamy-2, reddish-3, bright red-4), and scar formation (stiff-1, moderate-2, soft-3, none-4). The data are shown as modes from 6 rats from each group.
Burn tissues of the rats were observed for histology and histopathology. The results are summarized (shown in Table 2 and Fig. 4). After creating the burn wound on day 0, all groups showed epidermal degradation at the upper part of the ulcer. Additionally, the presence of edema at the lower part of the ulcer was evident in all groups. On day 7, all groups showed partial necrosis with epithelial loss and necrosis at the lower part of the ulcer. However, white blood cell (WBC) aggregation (score 1+) was profound at the lower part of the ulcer, which was similar in the NSS and blank hydrogel groups. However, the MLH and mupirocin ointment groups showed moderate WBC aggregation (score 2+). On day 14, at the lower part of the ulcer, all groups showed an absence of edema. The absence of necrosis showed in the NSS, blank, hydrogel, and MLH groups but not in the mupirocin ointment group. WBC aggregation was similar in the NSS, blank, hydrogel, and MLH groups, which showed profound aggregation (score 1+), but the mupirocin ointment group showed moderate aggregation (score 2+). The MLH group demonstrated less granulation tissue than the other groups. More fibrotic scarring appeared in the blank hydrogel group than in the other groups. On day 28, all groups showed complete epithelialization in the lower part of the ulcer without edema, necrosis, WBC aggregation, or granulation tissue. Moreover, fibrotic scarring appeared in each group as numerous bridges or septa with a fibrosis score of 2+.
| Day | Treatments | Upper part of ulcer (epidermis) | Lower part of ulcer (dermis, subcutaneous tissue, muscles) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Degeneration | Necrosis with epithelial loss | Re-epithelization | Edema* | Necrosis* | WBC aggregation | Granulation tissue | Fibrosis | ||||||||||||
| Partial | Total | Partial | Complete | A | P | A | P | 0 | 1 + | 2 + | 0 | 1 + | 2 + | 0 | 1 + | 2 + | |||
| 0 | NSS | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| Hydrogel | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| MLH | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| Mupirocin ointment | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| 7 | NSS | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||
| Hydrogel | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| MLH | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| Mupirocin ointment | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| 14 | NSS | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| Hydrogel | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| MLH | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| Mupirocin ointment | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| 28 | NSS | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||||||||||||
| Hydrogel | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| MLH | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
| Mupirocin ointment | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |||||||||||||
*A = Absence, P = Presence, WBC aggregation and granulation tissue (0 = Scanty, +1 = Profound, +2 = Moderate), Fibrosis (0 = None, +1 = Expansion, +2 = Bridging).
The representative images were obtained from at least 6 specimens. V: Blood vessel, H: Hair follicle, W: White blood cell aggregation, E: Epidermis, D: Dermis. (Color figure can be accessed in the online version.)
New blood vessels could be seen in all groups. Blood vessels appeared within the areas of regenerated skin in the blank, hydrogel, MLH, mupirocin ointment, and NSS groups. More hair follicle cells were observed in the MLH group than in the other groups (shown in Fig. 5).
(a) New hair follicle; blue arrows show the hair follicle. (b) Blood vessel formation in healing wounds; black arrows show blood vessel formation. And bar chart shows blood vessel density within areas of regenerated skin. Data represented in mean ± S.D., n = 6. (Color figure can be accessed in the online version.)
The MMP-2/TIMP-2 ratios in the post-burn full-thickness tissue on days 0, 7, 14, and 28 after treatment are shown in Fig. 6. The results showed that the lowest MMP-2/TIMP-2 ratios observed in MLH and blank hydrogel groups were significantly lower than the NSS and mupirocin ointment groups on days 7 and 28 (p < 0.05). However, all groups indicated low levels of the MMP-2/TIMP-2 ratio on day 28. The MMP-2/TIMP-2 ratios in the MLH and blank hydrogel treated groups were lower than the NSS and mupirocin ointment groups.
Data represented in mean ± S.D., n = 6. * p < 0.05.
The expression of TGF-β1 is shown in Fig. 6. The results showed that the TGF-β1 concentration had mainly increased on day 7 after burning in all groups. After 7 d, all groups had gently reduced TGF-β1 concentrations along the duration of the study, but after 14 and 28 d there was a significant decrease of TGF-β1. On day 7, the wounds that were treated with blank hydrogels had the lowest levels of TGF-β1, followed by MLH, NSS, and mupirocin ointment (p < 0.05). However, on day 14, the wounds treated with MLH had the lowest level of TGF-β1 compared with the other treatments. Finally, on day 28, the TGF-β1 concentrations in the MLH and blank hydrogel groups were less than in the other groups.
Immunohistochemical Analysis of Post-burn Full-Thickness Wounds after Treatment with MLHImmunohistochemistry was used to assess the number of CD3+, CD20+, and CD68+ cells (shown in Fig. 7). The staining of CD3+ and CD20+ cells was found petite cells as lymphocytes (shown in Fig. 7). On day 7, the CD3+ and CD20+ cells were gathered in the layer of skin and at the edges of the wounds. The CD3+ and CD20+ cells displayed similar behavior in each group. Both positive cells increased significantly and rendered peaks on day 7, and then decreased progressively until day 28. On day 7, the blank, hydrogel and MLH groups showed that the CD3+ and CD20+ cells were significantly higher than in the mupirocin ointment and NSS groups (p < 0.001). However, on day 14, the NSS group showed that the number of CD3+ cells at the edges of the wounds were lower than in the other groups. Moreover, the mupirocin group showed more CD20+ cells in the layer of skin than in the other groups. The number of CD20+ cells was significantly less than the CD3+ cells at each time point.
Data represented in mean ± S.D., n = 6. * p < 0.05.
The CD68+ cells were dispersed in the infiltrated cells on day 7 in all groups. On day 7, the CD68+ cells increased to the highest level in the blank hydrogel group, followed by the MLH, mupirocin ointment, and NSS groups. On day 14, the blank hydrogel group showed fewer CD68+ cells at the edges of the wounds than in the other groups. Nevertheless, the CD3+, CD20+, and CD68+ cells were concentrated in the deeper subcutaneous tissue on day 28.
Hydrogels have gained considerable attention due to their wide range of common pharmaceutical applications, especially in wound healing.19) The highlights of this study used complementary techniques to evaluate the safety and efficacy of a mupirocin nanoparticle-loaded hydrogel formulation in a wound healing application using cell lines and animal models. The hydrogel was the safest for skin cells, followed by MLH, mupirocin ointment, and pure mupirocin with no cutaneous toxicity to human epidermal keratinocytes, human fibroblasts, or macrophage-like cells. The macrophage-like cells (RAW264.7) play essential roles during the immune and inflammatory response, which occur during the inflammatory processes in healing.20) RAW264.7 macrophage cells produce NO, IL-1β, and TNF-α after an endotoxin challenge. Likewise, RAW264.7 macrophage cells suppress inflammation by producing IL-1β and TNF.21–24) The MLH and other samples induced an inflammatory response less than 100 pg/mL in the RAW264.7 cell line, which was non-toxic. Concentrations of IL-1β and TNF-α higher than 1200 ng mL−1 can indicate toxicity to keratinocytes.25) The MLH and hydrogel were neither toxic nor irritable to the skin, which demonstrated the safety of the cell lines in these studies. Moreover, IL-1β and TNF-α may play a role in migration.
The scratch assay represented the inflammation phase (second phase of wound healing) of keratinocyte or fibroblast proliferation and migration processes.18) Therefore, this study showed that the migration rates of MLH and hydrogel were higher than the other groups in the BJ cells such as fibroblast cells. Possibly the MLH and hydrogel might contribute to the wound healing in skin fibroblasts since Poloxamer 407 hydrogel was in the formulation.26) Stimulated fibroblast cell growth is a productive method for examining in vitro wound healing activity because the fibroblast cell growth, important information, relate to the migration, proliferation, and wound contraction in wound healing process.24) Whenever the cell monolayer is separated by scratching, it reacts to disruption of the cell-to-cell contacts by increasing of cytokines and growth factors concentration at the wound edge.27)
Assessment of wound healing in a rat burn wound model included a study of the wound morphology, wound contraction, histology of the wound, immunohistochemical analysis, and biomarker levels. The results showed that the wounds treated with hydrogel and MLH revealed better wound contraction than the mupirocin ointment. However, to understand the clinical significance of this finding, factors such as the degree of granulation, tissue proliferation, necrosis, and inflammation need to be quantified to explain wound healing.28) On day 14, the MLH group showed more improvement in burn wound healing than the hydrogel, mupirocin ointment, and NSS groups. Moreover, less wound secretion was observed in the hydrogel and MLH groups than in the mupirocin ointment and NSS groups. Wound secretion was normal during the inflammatory stage of wound healing. A minimal amount of secretion is considered normal wound drainage. However, a moderate to massive amount may indicate a high number of bacteria living on the wound or trauma to the wound.29) Previous studies reported that hydrogels can absorb and retain wound exudates.13)
The major physiopathology event of wound healing is the inflammatory response. An immunohistochemical analysis of the inflammatory profile, in particular the lymphocytes, evaluated the evolutional stages of wound healing.30) On day 7, this study showed that the number of CD68+ cells was higher than on the other days, which influenced the production of MMPs, IL-1β, and TGF-β by stimulating immune complexes.31) They are substances that stimulate fibroblasts to produce collagen and improve angiogenesis.32) The role of MMP-2 in lymph angiogenesis was considered in a modeling and theoretical study. MMP-2 degrades collagen I to switch to different patterning mechanisms for vascular endothelial growth factor.33) In addition, macrophages secrete collagenase to clean the wound and transforming growth factors to stimulate keratinocytes.31) This helps in the wound healing process.
In this study, the behaviors of CD3 and CD20 (T and B cells) were similar to previous reports in regard to wound healing.34) Previous studies used the immunohistochemical detection of CD3+ cells to identify T cells in mouse skin, which is an integral membrane protein widely expressed.35) On the other hand, immunohistochemical detection of CD20 indicates an activated-glycosylated phosphoprotein present on the membrane surface of B cells.36) The lymphocytes (T and B cells) infiltrated on day 7 that were likely correlated with the inflammatory react to active antigen in the peripheral lymphoid tissues that led to proliferation migration, and accumulation at the wound site. Possibly this occurred because the base formulation included the Poloxamer 407 hydrogel and the gelatin incorporated with mupirocin promoted wound healing.
Furthermore, the physicochemical properties of formulations (hydrogel and ointment) affect wound healing. In this study, we have designed MLH formulation containing Poloxamer 407 and cPVA hydrogel with a viscosity around 230–240 Poises at skin temperature and contained large amount of water (82.43%)37) whereas ointment has relatively higher viscosity and contained much less amount of water (10%).38) The viscosities and water content affected the wound healing by maintaining moisture at wound site, wound attachment and healing potential. Moreover, the P407-cPVA hydrogel has several functions to heal wounds because they can absorb and retain wound exudate, and repair skin function. Moreover, they also promote fibroblast proliferation and keratinocyte migration, which are both necessary for complete epithelialization of the wound. In addition, gelatin is the denatured form of collagen which is used as a scaffolding material for skin tissue engineering and wound care.36,39) Mupirocin nanoparticle gelatin carriers adhere to the surrounding tissues and have shown to support the formation of epidermal tissue. Poloxamer 407 increases cell growth and wound healing, which exhibits the early phase of wound healing related to the number of collagen fibrils.26) Hydrogels have received particular attention in wound dressings because of their properties such as the high water-holding capacity. Hydrogels also serve to moisten a wound site which can improve the wound healing process by preventing tissue dehydration and promoting accelerated angiogenesis.10–13)
In conclusion, MLH formulations were safe and effective on wound healing in cell lines and rat model. Hydrogel and mupirocin nanoparticles (gelatin as the carrier) in the MLH formulation had the greatest wound contraction, histological structures for wound healing, TGF-β1, MMP-2, and TIMP-2 concentrations, followed by mupirocin ointment. Lymphoid cells (CD3 and CD20), and macrophages (CD68) varied over the time of wound healing. They peaked on day 7 followed by progressive reduction until day 28. Therefore, this hydrogel is a promising formula for further development of biocompatible hydrogels loaded with antibiotics, such as mupirocin nanoparticles, for pharmaceutical products to ensure the safety of the participants in clinical trials.
We are grateful to the Drug Delivery System Excellent Center (DDESC), Faculty of Pharmaceutical Sciences, Prince of Songkla University for the research facilities.
This research was also supported by the Royal Golden Jubilee Ph.D. program of the Thailand Research Fund; PHD/0088/2559 for K. Sukanjana and National Research Council of Thailand (PHA610372S)
All authors were involved in the experimental studies.
Kamlungmak, Suwandecha, and Srichana designed the study.
Kamlungmak and Srichana were major contributors in collecting, analyzing, and interpreting the data.
All authors read and approved the final manuscript.
The authors declare no conflict of interest.
