The skin, being the biggest organ and covering the entire body, consists of two major tissue layers: a keratinized stratified squamous epithelium and dermis that are composed of two layers of connective tissue, including an interconnected mesh of elastin and collagenous fibers produced by fibroblasts [7,20]. Wounds on the skin are defined as physical, chemical, or thermal injuries that cause a loss of skin integrity. In the same way, the integrity loss of skin tissue can act backwards to initiate a complex set of events that finally lead to wound repair . In general, wound repair occurs in almost every type of tissue or organ to recover the damaged tissue and restore its defensive functions. Normally, wound healing is a complex biological process involving three highly interconnected and overlapping phases: inflammatory, proliferative, and remodeling phases [7,20]. Although wound healing is automatic in recovering skin tissue, in some cases the process leads to some disorders or trauma on the new recovering skin . Moreover, many drugs prescribed to enhance wound healing still suffer from low availability, high cost, and various detrimental side effects. Therefore, biologically active compounds of natural origin that are safe, reliable, clinically effective, of low cost, globally competitive, and better tolerated by patients are in great demand .
Crocodilians form an ancient reptile species and share their aquatic living environments with a variety of opportunistic pathogens and microbes. Although wounds or lesions resulting from fights with other crocodiles or different species occur frequently, they appear to heal rapidly and are almost infection-free, despite the harsh environment [9,16]. Previously, serum from Alligator mississippiensis blood has been intensively studied in terms of its biological properties. Therein, it was confirmed that crocodile serum possesses a wide range of antibacterial, amoebicidal, and antiviral activities, and its properties showed a much broader spectrum than that of human serum [12-14]. Likewise, the freshwater crocodilian Crocodylus siamensis has been intensively studied during efforts to conserve this critically endangered species, with a particular focus on investigations of its powerful immunity that includes the genomic DNA of immune cells and blood components . Serum as well as plasma of C. siamensis was reported to exhibit strong activity against Salmonella Typhi, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae [9,16,17]. Moreover, crocodile plasma and serum were found to directly affect bacterial cells through formation of blebs on the cell surface, subsequently leading to perturbation and damage of bacterial membranes [9,16,17]. Other reports further revealed potent antioxidant and anti-inflammatory activities of crocodile plasma and serum [8,15]. Although a possible influence has not yet been disclosed, anecdotal evidence suggests that C. siamensis serum and plasma might also exhibit a beneficial effect in wound healing processes. This is further supported by literature reports concluding that antimicrobial, antioxidant, and anti-inflammatory activities have a crucial influence on the progress of wound healing around the injured skin [1,3,11]. Therefore, to determine the role of C. siamensis serum and plasma during wound healing, the in vitro human kertinocyte cell line (HaCaT) was first focused on functional analysis regarding proliferative and migratory effects. Additionally, in vivo mouse excisional skin wound healing and wound infection healing models were used to study the cutaneous regeneration during skin tissue recovery.
Materials and Methods
Preparation of C. siamensis Serum and Plasma
Crocodiles (C. siamensis) were bred at the local Sriracha Moda Farm., Ltd., Chon Buri, Thailand. Blood samples were collected from the dorsal vein of adult crocodiles (1-3 years old) using a sterile syringe with needles (0.8 × 38 mm). The whole blood samples were transferred immediately into either a 1,000 ml bottle containing 0.08 g of EDTA for plasma collection or 15 ml conica tubes without any anticoagulants for serum collection. The blood samples were completely set or clotted by storing at 4℃ overnight. Plasma was collected from the liquid layer on the top of the blood samples, and clotting serum was obtained by centrifugation at 2,000 ×g for 10 min at 4℃. The crocodile plasma and serum were kept at -70℃ until required.
The human keratinocyte (HaCaT) cell line was kindly provided from Asst. Prof. Dr. Natsajee Nualkaew, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand. It was grown in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic:antimycotic (Gibco, USA) in a 5% CO2 humidified atmosphere at 37℃.
HaCaT cells (1 × 104 cells/well) were seeded into a 96-well plate and allowed to attach overnight in a 5% CO2 humidified atmosphere at 37℃. Then, the cells were incubated with crocodile plasma or serum at different concentrations (0-1,000 μg/ml). After that, 150 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide solution (MTT, 0.5 mg/ml) was added to each well and incubated for 4 h at 37 ℃. The reaction furnished a purple formazan product in the presence of viable mitochondria in living cells. After the medium was removed, cells were solved in 150 μl of DMSO, and the absorbance of formazan was detected at 550 nm in a microplate reader (UT-2100C, MRC, UK). Experiments were performed in triplicate. Cell viability was evaluated by comparing the respective absorbance of the experiment and the control.
In Vitro Scratch Wound Healing Assay
HaCaT cells were seeded into 96-well plates to a final cell density of 5 × 104 cells/well and cultured as a monolayer to confluence overnight. Scratch wounds were created using a sterile 200 μl pipette tip. Suspended cells were removed by washing twice with phosphate buffer saline (PBS). Then, the cell cultures were replaced immediately with fresh medium containing 1% FBS in the presence or absence of various concentrations of crocodile plasma (100, 150, nd 200 μg/ml) or serum (25, 50, and 100 μg/ml). Cells incubated in fresh medium containing 1% FBS were used as the control group. The samples were hen incubated for up to 48 h at 37℃ in 5% CO2. The same area of scratched wound edge was photographed at certain time intervals using an inverted microscope (Zeiss, USA) at three points per field. All experiments were independently carried out in triplicates. The area enclosed between the wound edges was measured using Image Pro plus 7.0 (MediaCybernetics, USA). The percentage of cell migration was calculated from the remaining gap size a t 3, 6 , 1 2, 2 4, 3 6, a nd 4 8 h a fter s cratch formation, compared with the initial gap size at 0 h.
In Vivo Mouse Excisional Skin Wound Healing Assay
Healthy ICR mice were purchased from the National Laboratory Animal Center, Mahidol University, Salaya, Nakhon Pathom, Thailand. The animals were housed at a temperature of 22 ± 1℃, relative humidity of 55 ± 5% and 12 h dark-light cycle. All experiments in this study complied with the “Guide for the Care and Use of Laboratory Animals.”
The experiments were performed according to a protocol previously published, with some modifications [1,3,7,20]. Twelve ICR mice were used. Before the wound was generated, the ICR mice were anesthetized with an intraperitoneal injection of sodium pentobarbital. The dorsal fur of the animals was shaved and the skin disinfected by swabbing with 70% ethanol cottons. On the depilated back of each mouse, circular wounds of 4 × 4 mm in average were generated by cutting carefully through the full thickness of the skin. The animals were randomly divided into four groups, including one untreated group and three treatment groups. Treatment was performed with crocodile plasma (60 μg/wound) or crocodile serum (60 μg/wound). Prednisolone (50 μg/wound) was used as a positive control. All treatments were performed during 12 days by daily application, and the progress of wound healing was observed and photographed every 3 days.
In Vivo Wound Infection Healing and Histological Assessment
Administration of crocodile plasma or serum on wound infections was performed as previously described [3,6], with slight modifications. Twelve ICR mice were randomly divided into four groups (n = 3 for each group) and full-thickness circular wounds of 4 × 4 mm in average were generated after sodium pentobarbital local anesthetic injection. Wounds were inoculated with 10 μl of the mid-log phase of S. aureus ATCC 25923 (1× 107 CFU/ml) in PBS. After 30 min of the infection, the wounds were treated individually with either crocodile plasma (2.67 × 103 μg/ml), serum (2.67 × 103μg/ml), or PBS used as the negative control and reapplied daily for 9 days. Each wound was photographed every 3 days. In addition, at days 3, 6, and 9 after wounding, mice were sacrificed (three animals in each group, two paired wounds per animal). The rectangular specimens of control and treated groups were taken out from the healed wounds for histological study. After that, tissue samples were fixed in 10% (v/v) formalin for 48 h. Then, the fixed tissues were cleared in xylene and embedded in paraffin. To determine the center of the wound and adequately monitor the healing process, the whole sample was serially cross-sectioned (7 μm) with a microtome and mounted on a glass slide. The sections were stained with hematoxylin and eosin. All images were observed under a light microscope and captured at 40× magnification.
The results of MTT assay and wound closure were calculated by using the analysis of variance. The differences between mean values of all data were compared using the least significant difference test. Statistical significance was set at p < 0.05.
Cell Viability and Proliferation
The possible cytotoxicity of C. siamensis plasma and serum were determined using the MTT assay. After cells were co-cultured with 0-1,000 μg/ml of plasma or serum for 24 h, no cytotoxic effects of the blood components on HaCaT cells were observed. Additionally, plasma and serum were found to promote the proliferation of the cells (Fig. 1). At a concentration of 3.9-1,000 μg/ml, C. siamensis plasma could greatly increase HaCaT cell proliferation within a range of 114-155% (Fig. 1A), whereas serum at the same concentration also led to a significant promotion of cell proliferation, ranging between 119% and 146% (Fig. 1B). Moreover, untreated HaCaT cells cultured in DMEM and supplemented with 10% FBS were used as a control and considered to represent 100% cell proliferation.
Fig. 1.MTT assay of C. siamensis plasma (A) and serum (B). HaCaT cells (1 × 104 cells/well) were cultured overnight in 96-well plates and then co-incubated with different concentrations of plasma or serum (0-1,000 μg/ml) for 24 h. Each result is expressed as the mean ± SD. Different letters (a-f) on the top of individual bars indicate statistically significant differences (p < 0.05) compared with untreated HaCaT cells.
Effect of C. siamensis Plasma and Serum on Scratch Wound Model
An in vitro HaCaT cell scratch assay was carried out to evaluate the effect of C. siamensis plasma and serum on keratinocyte spread and migration. The area enclosed by the edges of the wound monolayer was determined and expressed as the difference after wounding in progress of 0, 3, 6, 12, 24, 36, or 48 h (Figs. 2 and Fig. 3). As expected, the denuded region of wounds treated with plasma and serum was narrower than that of untreated wounds in a dose-dependent manner. Evidence for the efficacy of the investigated crocodile blood constituents could be clearly observed after treatment for 12h . Moreover, after treatment for 48 h, the majority of wound areas treated with the highest concentration of plasma and serum closed completely, whereas untreated areas did not show complete wound closure (Figs. 2A and 3A).
Fig. 2.Effect of C. siamensis plasma on in vitro scratched wound healing in the HaCaT cell assay. (A) The wound margin was photographed at 0, 3, 6, 12, 24, 36, and 48 h after wound scratching in the presence or absence of different concentrations of plasma (100, 150, and 200 μg/ml). (B) The percentage of cell migration signifies the remnant gap size at 3, 6, 12, 24, 36, and 48 h after making scratches, compared with the initial gap size at 0 h. Each volume is expressed as the mean ± SD. Different letters (a-c) on the top of individual bars indicate statistically significant differences (p < 0.05) compared with untreated scratched HaCaT cells.
Fig. 3.Effect of C. siamensis serum on in vitro scratched wound healing in the HaCaT cell assay. (A) The wound margin was photographed at 0, 3, 6, 12, 24, 36, and 48 h after wound scratching in the presence or absence of different concentrations of serum (25, 50, and 100 μg/ml). (B) The percentage of cell migration signifies the remnant gap size at 3, 6, 12, 24, 36, and 48 h after making scratches, compared with the initial gap size at 0 h. Each volume is expressed as the mean ± SD. Different letters (a-c) on the top of individual bars indicate statistically significant differences (p < 0.05) compared with untreated scratched HaCaT cells.
For scratched cells co-cultured with crocodile plasma, anecdotal tests revealed a significant difference of scratched wound closure in the presence of 200 μg/ml of plasma after 6 h with respect to the untreated control, whereas at concentrations of 100 and 150 μg/ml, slightly enhanced wound closures were observed after 24 h (Fig. 2B). Additionally, scratched cells co-cultured with 50 and 100 μg/ml of crocodile serum showed statistically significant differences after 6 h, whereas 25 μg/ml crocodile serum affected wound closure differences after 12h incubation (Fig. 3B).
Effect of C. siamensis Plasma and Serum on Mouse Excisional Skin Wound Model
The ability of C. siamensis plasma and serum to heal full-thickness skin wounds was evaluated by administration on mouse excisional skin wounds. As illustrated in Fig. 4, the size of the excisional skin wounds in the untreated group reduced slowly each day, whereas the size of skin wounds after treatment with plasma or serum decreased more rapidly and could be clearly observed after 9 days. Among plasma, serum, and prednisolone treatments, the healing sizes of the excisional skin wounds were not significantly different. Furthermore, almost complete wound closure was observed after 9 days, whereas the untreated wounds still displayed small amounts of edema. However, all mouse excisional skin wounds were completely closed after 12days.
Fig. 4.Effects of C. siamensis plasma and serum in the in vivo mouse excisional skin wound healing assay. The images of mice from each group were photographed every 3 days. The experiment was performed during 12 days by daily application of 60 μg/wound of plasma or serum, and 50 956;g/wound of prednisolone used as a positive control.
Effect of C. siamensis Plasma and Serum on in Vivo Wound Infection Model
In an attempt to assess whether C. siamensis plasma and serum could serve as a beneficial tool for wound infection repair, mid-logarithmic phase S. aureus ATCC 25923 (1 × 107 CFU/ml) was inoculated on mouse excisional skin wounds. The effect of plasma or serum treatments was assessed by gross examination and histological examination of epithelial gap closure and granulation tissue formation (Fig. 5). After 2.67 × 103 μg/ml crocodile plasma and serum were applied to the infected wounds, the uncovered area around the treated wounds decreased to a greater extent than the area treated with only PBS after 6 days. Moreover, after 9 days, complete closure of infected wounds could be observed in the plasma- and serum-treated groups, as well as the untreated group (uninfected wounds). In contrast, the infected wounds treated with PBS still showed small amounts of edema (Fig. 5A).
Fig. 5Effects of C. siamensis plasma and serum in the in vivo wound infection model. (A) Representative images of mice from each group were photographed at 40× magnification every 3 days using a light microscope. The experiment was performed during 9 days by daily application of either PBS, plasma, or serum at doses of 2.67 × 103 μg/ml. (B) Histology image of the epithelial gap of mice from each group. The edges of keratinocytes are indicated by arrows.
The effects of crocodile plasma and serum on infected wounds were further addressed using a histological method. The results indicate that re-epithelialization of the wounds treated with plasma was improved after 3 days. At day 6, an increase in the length of the wound edges of new migrating epithelium was clearly observed in plasma- or serum-treated wounds. At day 9, a similar increase was observed, as in most cases the epithelial edges had converged to completely cover the wounds after treatment with plasma or serum, whereas in the PBS-treated wounds the epithelial gap was still observed (Fig. 5B). In uninfected wounds (control group), the epithelium was completely in recovered and hair follicles were observed.
The process of wound repair begins with tissue repositioning by cell proliferation present in connective tissue . During proliferation and migration processes in epithelial formation, keratinocytes will migrate forward to cover the wound surface and fill the wound gaps, thereby leading to wound re-epithelialization and healing [3,20]. In order to elucidate the wound healing ability of C. siamensis plasma and serum, the first experiment focused on elucidating the possible cytotoxicity and proliferation on a human keratinocyte cell line, HaCaT. The data demonstrated that 3.9-1,000 μg/ml of plasma and serum showed no cytotoxic effects on HaCaT cells. Moreover, the blood components significantly promoted the proliferation of the cells up to 155% for plasma and 146% for serum. These observations led to the assumption that C. siamensis plasma and serum might possess pronounced wound healing properties related to enhanced cell proliferation and migration. Supporting evidence is provided by the observation that, depending on the season, crocodilians frequently engage fights with other crocodiles or other species, often resulting in severe wounds of their skin tissue. Interestingly, these injuries appear to heal rapidly and are almost infection-free, although these animals can carry a high burden of fecal coliforms from their aquatic environment [2,21]. Considering these facts, it appears plausible that plasma and serum from C. siamensis may contain a variety of substances with antimicrobial or wound healing properties.
The scratch wound healing model provided an experimental means to investigate the potential effects of C. siamensis plasma and serum on HaCaT cell migration. HaCaT keratinocyte migration is often assessed using monolayer cultures in a scratch assay [3,19,22,23]. Furthermore, keratinocytes represent a cell line that mimics many properties of normal epidermal keratinocytes, is not invasive, and can differentiate under appropriate experimental conditions. Moreover, the HaCaT keratinocyte exhibits a migration index similar to primary human keratinocytes . From the literature, several soluble factors such as transforming growth factor (TGF)-β and cytokines (i.e., Granulocyte macrophage-colony stimulating factor (GMCSF), Interleukin (IL)-1β, and IL-6) have been shown to influence keratinocyte migration . As expected, the current study indicated that after scratching HaCaT cells, C. siamensis plasma and serum were able to enhance the migration of the cells in a dose-dependent manner. Consistent with the in vitro results, a remarkable enhancement of wound repair was also observed in the mouse excisional skin wound model. The repair ability of plasma, serum, and prednisolone showed no differences for full-thickness skin wounds. Besides this, C. siamensis plasma and serum also resulted in enhanced healing of S. aureus ATCC 25923-infected wounds, which occured faster than in PBS-treated wounds. Consequently, wound infection of tissues was further addressed using histological methods, confirming the ability of C. siamensis plasma and serum to support the re-epithelialization of the wound on mouse skin. These findings support the abovementioned idea that plasma and serum from C. siamensis may contain antimicrobial or wound healing promoting substances that are capable of improving the recovery properties of wounds or injured skin tissue. Moreover, the previously reported antimicrobial activity of crocodile plasma and serum against S. Typhi, E. coli, S. aureus, S. epidermidis, K. pneumoniae, P. aeruginosa, and V. cholerae is likely to be responsible for an enhancement of wound healing by preventing pathogenic infections. This is further supported by reports about the wound healing promoting activity of antimicrobial compounds of the cathelicidin LL-37 family. Cathelicidin was demonstrated to function as the activator of HaCaT cell migration, which, in turn, was related to actin dynamics and associated with augmented tyrosine phosphorylation of proteins involved in focal adhesion complexes, such as focal adhesion kinase and paxillin. Moreover, the antimicrobial peptide could significantly improve reepithelialization and granulation tissue formation in the mouse model . The effect of plasma-enhanced wound healing is likely related to platelets suspended in the plasma. However, not only platelets aid in the clotting process, as myriads of growth factors and cytokines also promote the wound repair, including TGF-β, TGF-α, fibrinogen, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, platelet thromboplastin, thrombospondin, coagulation factors, calcium, serotonin, histamine, and hydrolytic enzymes [4,18].
During wound healing, inflammation is one of the crucial processes for the restoration of damaged sites. The inflammation phase begins immediately, and, after injury, platelets start first to aggregate and seal the bleeding by forming thrombi at the wounds . Consequently, the inflammatory process begins with vasoconstriction, which favors homeostasis and releases inflammation mediators, followed by epithelization, angiogenesis, and collagen deposition. Additionally, a variety of immune cells are attracted to the wound injury and secrete pro-inflammatory cytokines. The inflammatory cells, notably neutrophils, also produce large amounts of reactive oxygen species (ROS). At excess concentrations, ROS can cause cytotoxicity and induce damage to surrounding tissues, leading to delayed wound healing [1,7,10]. Therefore, there is a high possibility that C. siamensis plasma might enhance both in vitro and in vivo wound healing processes by its anti-inflammatory and antioxidant activities. As described previously by Phosri et al. , plasma from C. siamensis was found to scavenge ABTS and hydroxyl radicals, with the highest antioxidant activity of 57.3% inhibition in the hydroxyl radical assessment. In addition, crocodile plasma revealed anti-inflammatory activity by inhibition of nitric oxide production in the murine macrophage (RAW 264.7) model. Hence, all of our data corroborated the hypothesis that substances having anti-inflammatory and antioxidant properties bear a significant potential to serve as effective therapeutic substances to accelerate the wound healing process [1,7].
In conclusion, to the best of our knowledge, no reports covering the wound healing activity of C. siamensis plasma and serum have yet been disclosed. Here, we present the first report evaluating the ability of C. siamensis plasma and serum with respect to both in vitro and in vivo wound healing. Using the HaCaT human keratinocyte cell line scratch wound model, we were able to confirm that C. siamensis plasma and serum significantly enhanced cell proliferation and migration during wound closure. In the mouse excisional skin wound model coupled with S. aureus ATCC 25923 infection, the crocodile blood components also exhibited the ability to accelerate healing and further to promote re-epithelialization of the injured skin. Hence, crocodile plasma and serum with their innate wound healing properties could be utilized as a natural resource for the development of novel therapeutic products.