Background
Globally, burn injury is rated as the fourth frequent out of all injuries. Approximately 500,000 burn patients have to be treated in the USA each year [
1]. Despite recent advances in burn wound skin management, the death rates remain high throughout the world [
2] with a vast majority (eleven fold higher) in low-income countries [
3]. For instance, in 2003, about 173,000 children were burned in Bangladesh where burn is the fifth leading cause of childhood illness [
4]. However, in particular, peoples of rural areas are more prone to the burden of burn related mortalities and morbidities. Fast aid and swift treatment for burn patients are vital to increase the survivability by closuring and protecting the burn wounds as immediately as possible to mitigate disabilities and fatalities. In most of the developing countries, peoples would like to treat burn injuries immediately at home before going to a clinic [
3]. Nevertheless, there are still lacking of a low cost and effective fast aid product to manage of burn injuries in an efficient and rapid manner [
5].
Pathophysiologically, burn is considered one of the most severe types of wound as it is easily susceptible to infection due to vascular necrotic tissue and loosening of epidermal integrity [
6]. Healing of wounds is a dynamic process including various overlapping phases [
7] such as ‘early inflammatory phase’ that inhibits infection during healing as well as destroys necrotic tissue and triggers signals essential for wound repair [
8], the ‘proliferative phase’ involves wound closure and restoration of vascular network [
9] and finally the wound scar matures during the ‘wound remodeling phase’ [
10]. Nonetheless, burn wound healing is often interrupted by excess inflammation leading to delayed healing and increased pain. Furthermore, scar tissue formation, incomplete re-epithelialization and absence of complete collagen remodeling are also hindering issues of burn healing [
11,
12]. During skin wound healing two cell types, namely keratinocytes and fibroblasts, interact in the proliferative phase [
9]. By a feedback loop keratinocytes and fibroblasts increase cell proliferation rate and wound contraction vigorously [
13]. The immune cell- macrophages that stimulate keratinocytes and fibroblasts to release the factors for increasing angiogenesis, collagen production, and epithelialization [
7]. Due to having these features, keratinocytes based burn wound healing products such as single cell keratinocyte spray solution and keratinocyte cell sheet are available in the developed world [
13,
14]. In 2013, the possibility of amniotic fluid derived mesenchymal cells (AF-MSCs) as a source for cell based wound healing therapy has been reported [
15,
16]. But the usefulness of these highly sophisticated and costly therapeutic products remains out of affordability for third world people.
Human amniotic membrane graft is one of the most medically accepted and widely used biomaterials in burn wound healing treatment from 1910 on [
17,
18]. It acts as a scaffold for proliferation and differentiation of new epithelial cells due to presence of factors such as fibronectin, elastin, nidogen, collagen types I, III, IV, V, VI, and hyaluronic acid [
19‐
21]. Alongside lacking of histocompatibility antigens HLA-A, B and DR [
22], it possesses an anti-inflammatory effect [
23]. However, the processing, transportation and storage of intact thin sheet of amniotic membrane has limited clinical applications due to associated cost. In 2017, the Atala group reported that dissolved amniotic membrane with hyaluronic acid gel can speed up the skin wound healing process [
24]. Besides the human materials, plants extract are also experimented to have burn healing properties. For instance,
Aloe vera (AV) has been used in treating burn associated wound and observed to be effective in burn wound management [
25,
26]. Because of anti-inflammatory effects, AV is of high usefulness in the treatment of skin wounds and first to second degree burns [
27]. Additionally, AV treatment significantly increased the collagen synthesis and remodels collagen composition (type III) to promote wound healing, contraction and the breaking strength of resulting scar tissue [
28,
29]. Importantly, it has been demonstrated that AV has greater efficacy over silver sulfadiazine cream in the treatment of second-degree burns [
30,
31]. In the developed world, recombinant growth factors and cellular tissue-engineered skin substitutes-based wound treatments are available and clinically practiced [
32]. However, this sophisticated approach is associated with high costs for patients in the low-income countries [
33]. Some reported commercial skin grafts such as integra and biobrane are available which have been shown to improve wound healing but they are also expensive and sometimes do not deliver optimal outcomes [
34,
35]. Thus, there is a need for a wound healing product with high clinical efficiency, which can be used rapidly, but retains the activity of a biological treatment.
Clinically, amnion has been applied as a wound covering bioactive material to heal split thickness skin burn wounds as well as for children with partial-thickness facial burns [
36,
37]. From our experience, amniotic membrane as a graft for burn wounds enclosure in Bangladesh appears to be advantageous [
38]. But the limited number of membrane donors and the lack of trained personnel in amniotic graft processing are major challenges. Further, amniotic membrane grafting service is available only in city areas at a very limited scale. Other limitations including instant requirement of physicians to do the transplantation of the graft and the sheet of amnion is generally held in place with sutures or additional bandaging [
24]. Considering the described treatment limitations on the one side and the advantages of wound healing properties of human amnion and
Aloe vera on the other side; this study aimed to develop a novel cost efficient product which in fact should be easy to produce and to store, physiologically effective and which application does not require a medic. Thus, we prepared three novel gel products from the extract of amniotic membrane (AM),
Aloe vera (AV) and the combination (AM+AV) which were later characterized both in vitro and in vivo
. We have demonstrated the usefulness of AM, AV and AM+AV gels as wound healing biomaterials which can accelerate burn wound closure through contraction, re-epithelialization, reduced inflammation and increasing angiogenesis in an animal model for skin burn.
Methods
Ethical approval
The collection and use of cesarean sections derived amniotic membrane for research and grafting purpose was approved by the ethical committee of Atomic Energy Research Establishment, and permitted by the “Human Organ / Tissue Donation and Transplantation Act, 1999” Govt. of Bangladesh. Written consent from the amniotic membrane donor was taken for amniotic membrane collection for use in research purpose. The ethics committee of Jahangirnagar University recommended and approved the animal model (Wistar Rats) for this study of skin irritation, burn induction following ARRIVE guidelines. All efforts were made to prevent any unnecessary and harmful animal handling.
Collection of placenta and preparation of human amniotic membrane
Human placenta/amniotic sacs were collected during cesarean sections and kept in 4 °C. Within 24 h we processed and prepared the membrane as described before [
24,
38]. Amniotic membranes were separated carefully from chorionic membrane manually and washed with PBS (Gibco) repeatedly until a complete elimination of blood clots was achieved. After that, the whitish membranes were transferred into sterile petri dishes and frozen at subzero temperature overnight and freeze-dried (Alpha1-4LD, CHRIST, Germany) at − 55 °C for 24 h. Dried amniotic membranes were sterilized using gamma radiation at 10KGy with cobalt-60γ radiation sources. The membranes were then aseptically processed into powder form which was later used for gel preparation.
Fresh Aloe vera leaves were collected from the medicinal plant garden of NIB (National Institute of Biotechnology), Bangladesh by Rashedul Islam and Md Masud Rana. The plant Aloe vera were identified and collection of leaves kindly permitted by Md Moniruzzaman, Scientific Officer, NIB, Bangladesh. First of all, the leaves were washed with distilled water (DW) and wiped with 70% ethanol. The lower part of the leaves was cut to allow Aloe latex to be removed. After removal of latex, the leaves were taken into a laminar chamber and cut in equal pieces of about 4cm2. The leaves were merged in absolute alcohol for 5 min to further sterilize. Leaves were then peeled off and the juice was collected by scraping. Afterwards, the juice was poured into dishes and allowed to freeze; and finally freeze dried at − 55 °C for 48 h to obtain powder form. It was possible to extract 635 g juice from 1 kg of leaves which finally led to 8 g of powder.
From the dried amniotic membrane powder and Aloe vera powder, 2 gram of each sample was used for gel preparation. In total three types of gel formulations were prepared (i) AM (6% CMC-Na (Loba Chemie), 2% AM (2 g of amniotic membrane powder), 0.02% methyl paraben (SUPELCO-Sigma Aldrich), 5% glycerine (CP, China), 0.05% triethanol-amine (Merck), and DW up to 100 ml), (ii) AV (6% CMC-Na, 2% AV (2 g of Aloe vera powder), 0.02% methyl paraben, 5% glycerine, 0.05% triethanol-amine, and DW up to 100 ml), and (iii) AM+AV (6% CMC-Na, 1% AM (1 g of amniotic membrane powder), 1% AV (1 g of Aloe vera powder), 0.02% methyl paraben, 5% glycerine, 0.05% triethanol-amine, and DW up to 100 ml). The homogeneity of all formulated gels was confirmed by visual analysis. For assessing the pH of the different gel preparations, 2.5 g of each gel was dissolved in 25 ml of DW and incubated for 2 h. The measurements were done in triplicates and the average values were taken into consideration. The pH of these gels ranged from 6.5 to 6.9.
In vitro biocompatibility and cytotoxicity assay
In vitro biocompatibility and cytotoxicity test were done as described by Khan et al., (2012) [
39]. To do the heparinized human blood biocompatibility assay, AM, AV and AM+AV gels were diluted with different ratios of blood. A blood sample of the same donor diluted with DW and saline water (SW) at the same ratios as done for the gels was used as a control. After an incubation time of 2 h at room temperature, the blood/gel mixtures were spread on glass slides, and observed under a light microscope for possible morphological changes in the blood cells.
In vitro cytotoxicity tests of the AM, AV and AM+AV gels were performed using the brine shrimp (Artemia salina) lethality bioassay method. Artemia salina eggs were hatched in a 1 L conical flask, filled with sterile artificial sea water (pH = 8.5) and constant aeration for 48 h. After hatching, active nauplii free from egg shells were collected and used for the assay. All three gels were dissolved in artificial seawater at 2.0, 1.0, 0.75, 0.50, and 0.25 mg/mL concentration in petri dishes in which the active nauplii were inoculated. After overnight incubation, the viability of the nauplii was counted. Artificial sea water without additions served as negative control and 0.50 mg/mL of vincristine sulfate (Sigma) was considered as positive control.
Human skin cell biocompatibility tests were performed by exposing HaCaT cells (CLS Heidelberg, Germany) for 48 h in the specific culture medium containing the formulated gels at distinct concentrations. Cells were cultured in DMEM (Gibco/Life Technologies) with 10% Fetal Bovine Serum (FBS) (Gibco/Life Technologies) and 1% Penicillin/Streptomycin (Gibco) at 37 °C in 5%CO2. These tests resulted in an optimal gel concentration of 500 μg/mL.
In vitro cell attachment and proliferating assay
For cell attachment study, 50 thousand of human keratinocytes (HaCaT) and human fibroblast (HFF1 cell line (ATCC; SCRC-1041)) cells were seeded in 2 ml media containing the previously prepared gels at a concentration of 500 μg/mL. The cells were allowed to attach to the culture dish undisturbed for 2, 4, 6, and 8 h in case of HFF1; for 3, 6, 9 and 12 h in case of HaCaT, respectively. At each time interval microscopic images were taken to evaluate the attachment rate of the cells in the different conditions. To assess the proliferation of HaCaT and HFF1, equal numbers of cells were expanded on plates in the media containing AM, AV and AM+AV gels. The images were taken from day 2 to day 6 to visualize the proliferation of the cells. Media were replaced every other day.
In vitro wound healing scratch assay
The scratch assay was performed as described by D’Agostino and co-workers to study cell migration and to determine the time period required for wound closure in vitro [
40] in presence of the three gel formulations (500 mg/mL). When cells attained 95–100% confluency, HaCaT and HFF1 were serum starved for 24 h before initiation of the scratch wound. Scratch wounds were created in confluent cell monolayers using a sterile p200 pipette tip ensuring that each wound had the same dimensions. After that, the cells which were detached by this process were removed from the culture dish by three times washing with PBS. Cell migration and wound closure were observed at 0 h, 18 h and 30 h and images were taken by light microscopy.
In vivo irritability study
To assess in vivo irritability and applicability of the gels, the dorsal skin hairs of female Wistar rats were shaved on the date of experiment [
41]. Total 12 animals were experimented and randomly were assigned to three groups (AM, AV and AM+AV). The animals were treated with 1 ml gel daily up to 7 days and finally the treated skin was visually examined for erythema and edema.
Rat model for artificial burn induction, re-epithelization and wound contraction
In total 40 healthy female Wistar rats of 180–200 g body weight were used in this study and randomly assigned into four experimental groups (control/no gel, treated with AV, treated with AM and treated with AM+AV). All animals received human care according to the guideline for the care and use of laboratory animals published by NIH. Rats were fed a standard rat chow and tap water ad libitum. The rats were kept in the animal quarter at a temperature of 25 ± 2 °C, humidity 50–55% and with 14 h light/10 h dark cycles. Each rat was anesthetized with Ketamine HCl solution (Gonoshasthaya Pharmaceuticals Ltd., Bangladesh) of 100 mg/Kg body weight by intra peritoneal injection. Subsequently, the hair at dorsal region was trimmed using electric hair clipper and then shaved with sharp blade. The shaved areas were cleansed with alcohol swab.
Burns were created using a piece of aluminum (981.875mm
2) heated to 100 °C for 5mins which was applied for 15 s on the shaved area of rats [
42]. The animals were treated with 1 ml of the distinct gel on daily basis, topically, for a period of 30 days. Re-epithelization was monitored by recording the number of days required for crust to fall away, leaving no raw wound behind [
43]. To monitor wound contraction, progressive changes in wound area were measured. Using the formula [
42] below, the percentage of wound contraction was calculated on the respective day.
$$ \%\kern0.5em Wound\kern0.5em Contraction=\left(\left( Initial\kern0.5em Wound\kern0.5em Size- Final\kern0.5em Wound\kern0.5em Size\right)/ Initial\kern0.5em Wound\kern0.5em Size\right)\times 100 $$
At the specific days, the gel treated and non-treated animals were anesthetized by intraperitoneally administration of 100 mg/kg ketamine, skin tissue/biopsy were taken and finally rats were sacrificed by cervical dislocation. Skin tissues were collected for histopathological analysis.
Histology of skin: hematoxylin and eosin (H&E) staining
Skin specimens from each group were collected on the 6th, 12th, 18th, 24th, and 30th day after burn induction. Skin biopsies were embedded in paraffin blocks after overnight fixation in 10% formal saline solution. Embedded skin tissues were cut into sections of 5 μm thickness using a microtome (Leica, RM 2125 RTS, USA) and collected on glass slides. Afterwards, the sections were deparaffinized and stained with H&E. The stained histological sections were examined and evaluated in random order. Images were taken with Optika B-350 light microscope. A score of 0–3 was given to each section according to presence of inflammatory cells and levels of angiogenesis and epithelialization as previously described by Sedighi et al.
, 2016 [
44] and Kulac et al.
, 2013 [
45] with minimal modifications (Table
1).
Table 1
Histological scoring parameter of epithelialization, angiogenesis, granulation tissue formation, and inflammatory cells
Inflammatory cells | 1–5 inflammatory cells per histological field | 5–8 inflammatory cells per histological field | 8–11 inflammatory cells per histological field | 11–15 inflammatory cells per histological field |
Epithelialization | Absence of epithelial proliferation in ≥70% of tissue | Incomplete epidermal organization in ≥50% of tissue | Moderate epithelial proliferation in ≥60% of tissue | Complete epidermal remodeling in ≥80% of tissue |
Angiogenesis | Absence of angiogenesis including congestion and hemorrhage | 2–4 vessel per site, congestion and hemorrhage | 4–6 vessel per site, slight congestion | 7–8 vessel per site vertically disposed towards the epithelial surface |
Granulation Tissue | None, completely disorganized and distorted | Minimal/immature thin | Mild/moderately mature granule layer | Evident/ Thick, ≥80% organized |
Statistical analysis
All statistical analyses were calculated by one way independent test using SPSS (SPSS version 22.0, SPSS Inc., Chicago, IL, USA). All quantitative data were presented in this study including mean (±) standard deviations (SD). P < 0.05 was considered as statistically significant.
Discussion
Beside active ingredients, the applicability of burn wound healing gel is dependent on the properties such as pH, appearance, homogeneity, and viscosity. Carbopol is a widely used gelling agent for producing burn-healing gels for skin application. CMC-Na salt is another agent used alongside with carbopol [
46]. However, in this study the gel was prepared from human amniotic membrane extract (AM),
Aloe vera extracts (AV), and a combination of both AM+AV using 6% CMC-Na salt as a gelling agent. Extensive studies confirm the effectiveness and safety of lyophilized amnion as a wound dressing and grafting materials for promoting the healing process and preventing infections [
47]. Khorasani et al.
, (2009) reported that AV cream or gel could be more effective than silver sulfadiazine cream in treating burn wound healing [
30]. The end products (gels) in all three conditions evaluated in this study were homogenous, granule less and of a whitish creamy color (Fig.
1a10, 1b3, 1c). In addition, pH of all formulations ranged between 6.5–6.9, and this pH does not interfere with skin physiology [
48]. Erythema and edema are the common symptoms of skin irritation which lasts for three to 7 days [
49]. Our formulated gels did not induce any irritation including no erythema or edema on rat skin upon topical application for a period of 7 days (Fig.
4c). Thus, CMC-Na salt, AM and AV extract formulated gel could be useful as wound healing gel as the gels have good spreadability and consistency. In vitro and in vivo studies clearly and collectively demonstrated the potentials of the formulated gels from amnion when combined with
Aloe vera. In this preliminary research, we found that the formulated gels can induce and accelerate the proliferation and attachment of HaCaT and HFF1 cells (Fig.
3), and promote wound healing in vitro (Fig.
4a, b) [
50]. Amnion has been reported to facilitate the migration of epithelial cells, reinforces attachment, and promotes proliferation [
51].
Macroscopic morphological analysis demonstrated the gel-based acceleration of re-epithelialization and wound contraction in vivo (Fig.
5a, b, c). Microscopic observation of AM+AV gel treated tissue sections allowed us to appreciate the effectiveness of the formulated gel in regard to epidermis and dermis formation and the thickness of epidermis (Fig.
6a). In present study, we observed that upon completion of wound healing few scar tissues remained in all treated rats. Scar formation in all kinds of wound healing are normal [
52] and exist even after complete healing. However, the AM treated experimental group had the lowest scar formation. Regardless of the variation in treatment procedures, second degree burn required 25–35 days to heal completely [
53]. Due to presence of anti-inflammatory characteristics [
23,
44], amniotic gel treated rats had lower inflammation than AV and AM+AV (Fig.
6c). Inflammation is an early stage event for burn healing which should be decreased within a certain healing period [
54]. During wound healing, at early phases inflammatory cells increased but in later stages decreased gradually due to granulation, the formation of new capillaries, and deposition of collagen [
2]. Excluding collagen level determination, other incidences are similar to our study [
55]. Histological analysis revealed that the AM+AV group had a higher epithelialization rate apart from inflammation (Fig.
6a19, 6b). Wound healing is related to wound contraction and wound re-epithelialization [
56] which has been shown for gel treatment in a mouse model [
50].
Angiogenesis is another important event in burn healing where endothelial cells’ proliferation rate in the wound area is rapidly increased after burning to form blood vessels. Hamid and Soliman (2015) reported that AV can increase angiogenesis [
2] for a better supply with nutrients and oxygen because of acemannan in AV [
57]. Histologically, AM, AV and AM+AV treated wounds had increased numbers of blood vessels, particularly small and newly formed (Fig.
6a17-a19, 6d). Besides supporting in vitro keratinocyte proliferation, we also observed some proliferating basal keratinocytes in vivo residing in the intermediate zone of the epidermis and dermis (Fig.
6a19). These properties of the tested gels could be explained by the presence of stimulatory factors in amniotic membrane which also been supported from the results of Murphy et al. (2017) [
24].
Amniotic membrane has been reported to provide a niche for the cells to adhere, grow, proliferate, migrate and differentiate, and could possibly contribute to the production of angiogenic micro-environment indirectly which allows AM to improve burn healing [
58]. We found that a combination of both AM and AV synergistically improved epithelialization. On day 30, epithelialization profile was significantly higher in the AM+AV group. Amniotic membrane is composed of collagen type IV, V and VII which promotes growth of epithelial cells, facilitates epithelial cell migration, strengthens basal epithelial cell adhesion, promotes differentiation of epithelial cell, and prevents apoptotic cell death [
59]. In principal, we have prepared gels from medically discarded materials, at low cost that provides excellent burn wound coverage. These formulated gels showed potential to be used as fast aid ointment in burn wound management. Although we demonstrated significant improvement of burn wound healing in rats treated with AM+AV gel, however, some limitations are associated with this animal model such as the remarkable native regeneration potential of the rat skin. For future application, it would be crucial to identify the key factors in the amnion that are responsible for the acceleration of the wound healing process.
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