The Effects of Aloe vera on Wound Healing in Cell Proliferation, Migration, and Viability

Introduction. Aloe vera is sometimes used as a folk remedy for minor wounds and burns, but its mechanisms of action in wound healing are unclear. Objective. In this study, the authors evaluate the effects of A vera on wound healing. Materials and Methods. In vitro analyses of cell proliferation and migration were conducted on normal human primary skin fibroblasts and keratinocytes in growth media with A vera solution and preservatives at various concentrations. Growth media with preservatives but without A vera solution served as the control. Results. Aloe vera had significant stimulatory effects on cell proliferation and migration of both fibroblasts and keratinocytes. Surprisingly, A vera also exhibited strong protective effects on preservative-induced keratinocyte death. Keratinocytes in the growth media with both the preservatives and A vera had dramatically higher viability than cells in the control media without A vera. Conclusions. The results suggest A vera accelerates wound healing by promoting the proliferation and migration of fibroblasts and keratinocytes and by protecting keratinocytes from preservative-induced death.

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Wounds and related injuries remain a major cause of death and disability. Wound healing is a complex, highly regulated process that includes cellular, molecular, biochemical, and physiological events that permit living organisms to repair accidental lesions. This process includes 3 overlapping phases: inflammation, proliferation and tissue formation, and tissue remodeling.¹ These events are initiated at the time of physical injury and continue throughout the healing process.²

The proliferative phase involves reepithelialization and granulation tissue formation, which includes fibroplasia and angiogenesis. Reepithelialization refers to the resurfacing of the epidermis by keratinocytes, the main cell type of the skin epidermis, from the wound edges and/or residuals of skin appendages.^1,3 Keratinocytes begin migration 12 to 24 hours after injury. The migration and proliferation of these cells are key events for reepithelialization and closure of the wound gap. During granulation tissue formation, fibroblasts migrate, proliferate, and synthesize large amounts of collagen and other extracellular matrix to fill the dermal defect in a process known as fibroplasia.⁴ During angiogenesis, new blood vessels are formed in the wounded area. Angiogenesis depends on the migration and proliferation of endothelial cells from pre-existing blood vessels in the wound edge.^1,5

The objective of wound management is to heal wounds in the shortest amount of time with minimal pain, discomfort, and scarring.⁶ Thus, improving treatment for wound healing and tissue repair will improve the quality of life of patients with wounds as well as reduce the overall cost of wound-related health care.

Aloe vera is the name often used for A vera Linne or A barbadensis Miller. This plant has more than 400 identified species and belongs to the Aloeacea or Liliaceae family.⁷ The A vera leaf contains chemical compounds (ie, acetylated mannans, polymannans, anthraquinone C-glycosides, anthrones, anthraquinones, and lectins)^8,9 and has been traditionally used in many cultures for its therapeutic properties. The mucilaginous gel from the leaf pulp of A vera has been incorporated into many cosmetic and alternative medicines for rejuvenation, wound healing, and other dermatologic conditions.¹⁰ Despite its wide use as a folk remedy, few scientific studies have been conducted regarding the physiological function of A vera in wound repair.^11-18

Previous studies suggested A vera, or 1 or more of its constituents, promotes wound healing in various animal models^11-16; however, its mechanism of action remains unclear.¹¹ Chithra et al¹⁵ evaluated the effect of A vera gel on full-thickness wounds in diabetic rats. Their results¹⁵ indicated A vera treatment may enhance the process of wound healing by affecting fibroplasia, collagen synthesis, and wound contraction.

In a recent study by Moriyama et al,¹⁷ the authors showed A vera promoted keratinocyte proliferation and migration in vitro and improved the process of wound healing in an ex vivo assay. Feily and Namazi¹⁹ conducted a review to evaluate the efficacy of A vera preparations on the treatment of skin diseases using an in vivo murine model and clinical studies. Their murine model¹⁹ found oral A vera preparation was effective for wound healing; however, their clinical studies¹⁹ found topical A vera had no preventive or protective effects on skin injuries due to radiation, sunburn, or suntan but was effective in the treatment of frostbite and burn wounds. In addition, Feily and Namazi¹⁹ found A vera had antimicrobial and antifungal effects.

The efficacy of topical A vera in burn wounds was evaluated in a systematic review by Maenthaisong et al.²⁰ They included 4 clinical studies with 371 patients. These studies compared time of healing, success rate of wound healing, and rate of reepithelialization of the topical application of A vera against that of standard gauze, framycetin, and silver sulfadiazine in burn patients.²⁰Aloe vera was found to accelerate the rate of reepithelialization and improve wound healing times for burn wounds; the authors concluded A vera may be an effective therapy for first- and second-degree burns.²⁰

However, there have been studies^18,21 with controversial results. Topman et al¹⁸ found A vera did not induce a significant effect on the migration kinematics of cultured fibroblasts. In 2012, a review by Dat et al²¹ of 347 patients from 7 studies evaluated the effects of A vera-derived products on acute and chronic wound healing. Three trials of patients with first- to second-degree burns compared the time of healing of A vera cream or mucilage (glue-like material of A vera leaf pulp) against that of framycetin cream (aminoglycoside antibiotic) or silver sulfadiazine cream. The results were contradictory; 2 trials showed A vera shortened wound healing time while the third showed no statistically significant effect.²¹ Yet, the trials were considered to have poor methodologies and a high risk of bias. In 3 other trials, the authors²¹ also found contradictory results when A vera cream or A vera-derived products were evaluated on postsurgical, postbiopsy, and pressure ulcer wounds against silver sulfadiazine cream. Thereby, they²¹ concluded cumulative evidence is insufficient to support topical A vera as a treatment for acute or chronic wounds, likely due to a lack of high-quality studies. They suggested more research on A vera is needed to determine its effectiveness on wound healing.²¹

There is a major gap in knowledge regarding the mechanisms of A vera treatments at the cellular level. To explore the role of this remedy in wound healing, this study evaluated the effects of the A vera plant on wound healing in in vitro models of human skin fibroblasts and keratinocytes, specifically in cell proliferation and migration.

Plant preparation

Whole leaf A vera liquid was obtained from Coats Aloe International Inc (Dallas, TX). The liquid contains 90% A vera (inner gel), deionized water, and preservatives made of 0.1% sodium benzoate, 0.1% potassium sorbate, and 0.14% phosphoric acid. Various concentrations of A vera solutions, at 1%, 2%, and 3%, were prepared.

For fibroblasts, the liquid was diluted in Dulbecco’s modified eagle medium (DMEM; Mediatech, Herndon, VA) with 2% fetal bovine serum (FBS; HyClone Inc, Logan, UT). For keratinocytes, the liquid was diluted in keratinocyte growth medium (EpiLife Growth Medium; Cascade Biologics, Portland, OR). In addition, since the A vera liquid contained preservatives (mixed with A vera product), the same growth media with preservatives but without A vera were included as controls (Ctr; 1% Ctr, 2% Ctr, or 3% Ctr, which refers to the relative preservative concentrations respectively) in the studies to ensure A vera was the only variable affecting the experiment.

Cells and cell cultures

Normal human primary epidermal keratinocytes and dermal fibroblasts were isolated from normal human neonatal foreskin. The Institutional Review Board of the University of Miami (Miami, FL) approved the protocol. The tissue samples were cut into strips, 3 mm to 5 mm in size, and incubated at 4°C overnight in Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA) with an addition of dispase (Sigma-Aldrich, St Louis, MO) at a concentration of 250 U/mL as described previously.^22,23 After incubation, the dermis was separated from the epidermis using forceps.

The epidermis was incubated in 5 mL of 1x Trypsin-EDTA (0.05% trypsin and 0.02% EDTA) for 15 minutes in a 37°C water bath and neutralized in 5 mL of keratinocyte growth medium with 20% FBS. The cells were then spun at 1000 rpm for 5 minutes at 5°C, resuspended in the keratinocyte growth medium, and plated in culture dishes. Keratinocytes were maintained in keratinocyte growth medium, supplemented with human keratinocyte growth supplements (HKGS; Cascade Biologics) at concentrations of 0.2% v/v of bovine pituitary extract, 5 µg/mL bovine insulin, 0.18 µg/mL hydrocortisone, 5 µg/mL bovine transferrin, 0.2 ng/mL human epidermal growth factor (EGF) plus antibiotic-antimycotic (Mediatech, Manassas, VA) with penicillin at 100 µg/mL, streptomycin at 100 µg/mL, and amphotericin B at 0.25 µg/mL. The primary cells were grown in a tissue culture incubator at 37°C and 5% CO₂. Cells from 5 to 6 donors were pooled at passage 1 and used for the study at passages 6 to 8.^22,23

Following the method described by Normand and Karasek,²³ the dermal sheets were further cut into 1-mm² to 2-mm² pieces and placed on the bottom of a 60-mm diameter tissue culture dish (Corning Inc, Corning, NY) and maintained in DMEM with 10% FBS and antibiotic-antimycotic with 100 µg/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B at 37°C in a 10% CO₂-humidified tissue culture incubator. Once outgrowing fibroblasts from the explants were 80% to 90% confluent (passage 0), they were subcultured in tissue culture dishes. Fibroblasts from 5 to 6 donors were pooled at passage 1. For the experiments, cells from 8 to 10 passages were used.^22,23

Cell proliferation assay

Primary human fibroblasts (1.18 × 10⁴ cells/mL/well) were plated in 24-well tissue culture plates in DMEM with 2% FBS and incubated overnight at 37°C and 10% CO₂. Cells were divided into 4 groups and treated with 2% and 3% A vera, and their respective Ctrs. Cells received fresh medium and treatment every 24 hours from day 0 to day 5 of the experiment. After the change of media, microscopic observation was performed daily prior to cell count. At days 1, 2, 3, and 5 of the study, fibroblasts were treated with 0.05% trypsin, detached from the dishes, and then counted using the dye-exclusion hemocytometer method. A high cell number indicated a strong effect on cell proliferation.

For keratinocyte proliferation assay, keratinocytes were plated in 24-well culture plates, 1.18 × 10⁴ cells/mL/well, in keratinocyte growth medium and treated with 1%, 2%, and 3% A vera along with their respective Ctr media. Cells were counted on days 1, 3, and 5 of the study with the same method as described for fibroblasts. Each treatment was done in triplicate, and each experiment was repeated at least twice.

Cell migration assay

The cell scratch assay, an in vitro wound model that correlates with in vivo incisional wound model,²⁴ was used to study the effects of A vera on fibroblast and keratinocyte migration. The cells were grown in 12-well tissue culture plates with designated wells for each concentration and corresponding Ctr groups. Once the cells were 95% to 100% confluent, a scratch (inflicted wound gap or no cell zone) was administered to each well. The vertical and horizontal cross-shaped scratch was made using a 200-µL pipette tip. The center of the cross, where the 2 scratch lines meet, was used to position the center of the wound gap. The wells were washed twice with Dulbecco’s phosphate buffered saline without Ca²⁺ or Mg²⁺ to clear any detached cells and then refilled with the appropriate treatment media with addition of mitomycin (Sigma-Aldrich) at 10 µg/mL to block cell proliferation.

Cell migration (gap filling) was examined and recorded using a Zeiss Axiovert 200 Microscope with Zeiss AxioCam imaging system (Carl Zeiss MicroImaging, Inc, Thornwood, NY) at hour 0 (immediately after scratch) and 24 hours after wounding. Gap areas were measured with Zeiss Axiovision V.4.1 Software (Carl Zeiss MicroImaging, Inc) and cell migration was quantified. The percentage of gap filled (PGF) was calculated using the Formula.

Percentage of gap filled then was graphed as a function of time elapsed versus PGF to show cell migration. A high PGF value indicated a strong effect on cell migration. The data were analyzed using GraphPad Prism v.5 Software (GraphPad Software, Inc, La Jolla, CA).

After wound creation, fibroblasts were divided into 2 groups. Group 1 cells were treated once daily with 3% A vera in DMEM with 2% FBS (3% A vera); group 2 cells were treated with the Ctr (3% Ctr). Keratinocytes were divided into 2 groups and treated with 1% A vera in keratinocyte growth medium and its Ctr (1% Ctr). Each treatment was done in triplicate, and each experiment was repeated at least twice.

Cell viability assay

Due to the results of the proliferation assay, a keratinocyte viability assay was performed to evaluate keratinocyte viability after treatment with A vera solution versus Ctr medium. Keratinocytes were plated in 24-well culture plates, 1.18 × 10⁴ cells/mL/well, in keratinocyte growth medium. Aloe vera media at concentrations of 1%, 2%, and 3% were used along with their respective Ctrs. Keratinocyte viability was recorded on study days 1, 3, and 5 using a Beckman Coulter Automatic Vi-cell cell viability analyzer (Beckman Coulter, Indianapolis, IN). Each treatment was done in triplicate, and each experiment was repeated at least twice.

Statistical analysis

The same software used for data analysis described in “Cell migration assay” was used for statistical analysis. Data were analyzed using 1-way analysis of variance (ANOVA) followed by an unpaired 2-tailed Student’s t test. A value of P ≤ .05 was considered significant.

Aloe vera strongly stimulated fibroblast proliferation

The effect of A vera on fibroblast proliferation was evaluated in a 5-day timing course. As shown in Figure 1, 2% concentrations of A vera solution showed strong stimulatory effects on fibroblast proliferation compared with the 2% Ctr media (P < .05 on day 1, P < .01 on days 2 and 3, and P < .001 on day 5). Concentration of A vera at 3% also showed a promising effect on cell proliferation starting at study day 2 (P < .05 on day 2, P < .01 on day 3, and P < .001 on day 5). Aloe vera at 2% and 3% concentrations performed without significant difference except on day 1, where 2% A vera was more effective than 3% A vera (P < .05).

Aloe vera stimulated fibroblast migration

The effects of A vera at the experimental concentrations on fibroblast migration are summarized in Figure 2. Fibroblasts treated with 3% A vera solution experienced accelerated gap filling (29%) compared with the 3% Ctr medium (17%) at 24 hours (P < .05).

Aloe vera strongly stimulated keratinocyte proliferation

The effect of A vera on keratinocyte proliferation was evaluated in a 5-day timing course. Throughout the study, both 1% and 2% concentrations of A vera demonstrated very strong stimulatory effects on keratinocyte proliferation compared with their Ctrs, with a P < .01 or P < .001 in all 3 days examined (Figure 3A). Aloe vera at 1% had better effects than A vera at 2% throughout the study, and A vera at 3% did not show significant effects.

Aloe vera increased keratinocyte viability

Due to the significantly lower mean cell count of keratinocytes as the concentration of A vera increased in the cell proliferation study, keratinocyte viability also was examined using a cell viability analyzer (Figure 3B). The results showed that A vera beneficially impacted viability throughout the entire experiment. Keratinocytes in 1% A vera showed a higher viability percentage throughout the experiment compared with the 1% Ctr medium (P < .01). This effect also was seen with 2% and 3% A vera solutions compared with the respective Ctr media (P < .001). For 2% and 3% Ctr media, cell viability readings were already near 0% by day 1; for 1% Ctr medium, viability markedly decreased from day 1 to day 5. Between A vera solutions, 1% and 2% A vera showed better effects on keratinocyte viability than 3% A vera during the experiment (P < .05). Furthermore, 1% A vera demonstrated more positive effects than 2% A vera on days 3 and 5 of the study (P < .01).

Aloe vera stimulated keratinocyte migration

The 1% A vera exhibited stimulatory effects on keratinocyte migration as summarized in Figure 4. The results of the keratinocyte scratch migration assay demonstrated that A vera at 1% concentration had a better effect on cell migration measured by PGF 24 hours after wounding compared with the 1% Ctr medium (P < .05).

Alternative and traditional medicines historically have been used for disease prevention and treatment. Traditionally, A vera has been used for wound healing for its anti-inflammatory, antiviral, and antiseptic effects.¹⁰ Most evidence supporting its potential benefits on the treatment of wounds comes from animal studies,^12-17 but its mechanism of action at the cellular level is still unclear. There is an absolute need to explore the scientific effects and mechanisms of action of novel and effective alternative treatments such as A vera.

In the present study, A vera showed strong promotional effects on fibroblast and keratinocyte proliferation (> 2-fold) and moderate (< 2-fold) but significant stimulatory effects on cell migration compared with the Ctr. Takahashi et al²⁵ demonstrated that A vera gel extract (AGE) alone and liposomal AGE positively stimulated fibroblast and keratinocyte proliferation in an in vitro analysis. In addition, A vera promoted keratinocyte proliferation and migration in a study conducted by Moriyama et al¹⁷ using in vitro proliferation and scratch migration assays. The authors¹⁷ also showed A vera enhanced epidermal development and keratinocyte migration during wound healing using a human skin epidermal equivalent model (ex vivo). Both results^17,25 are in concordance with the study reported herein.

Importantly, the present study revealed the strong protective effects of A vera on preservative-induced cell death. The results of the viability test showed that in the higher concentrations of preservative Ctr media (2% Ctr and 3% Ctr), all cells were dead within a 24-hour time period, suggesting the preservatives might be toxic to the keratinocytes. Even with a low concentration of Ctr medium (1% Ctr), the cells’ viability dropped throughout the experiment. Aloe vera at 1% to 3% concentrations showed beneficial effects in terms of cell viability. In the 3% A vera group, keratinocytes at day 1 had high viability that decreased gradually throughout the 5-day timing course. This effect was probably due to the protective effect of A vera over the toxic effect of the preservatives, which diminished in potency over the course of the experiment. The higher viability of keratinocytes treated with 1% A vera at day 5 suggests that A vera can counter preservative-induced cell death even at a low concentration, exhibiting a protective effect (Figure 3B). This also could be significant for protection against other chemicals or environmental pollution.

Although this study demonstrated potential mechanisms of A vera in promoting wound healing in cell proliferation and migration, in vitro wound healing assays cannot mimic the complexity of the conditions that take place during the in vivo wound healing process. Thereby, data obtained from in vitro assays should not be considered definitive and should be corroborated with in vivo models. In addition, this study discovered that A vera potentially has protective effects against preservative-induced cell death. As the protective effects of A vera have not been examined in vivo or with other chemicals, the relevance of this finding for in vivo toxicology is still unclear. Furthermore, toxicity-related cell death could be either apoptotic, autophagic, or necrotic cell death. More studies are necessary to understand the potential mechanisms in order to develop better therapeutic strategies for clinical treatment and protections.

The results of this study suggest A vera accelerated wound healing by strongly promoting fibroblast and keratinocyte proliferation and moderately stimulating cell migration. Surprisingly, A vera also shows protective effects against preservative-induced death of keratinocytes. These protective effects of A vera have not been previously described and may explain some of the positive effects of A vera for treating wounds. However, further studies need to be conducted in order to identify which components of the A vera plant aid in the wound healing process. These studies could lead to the production of specific treatment regimens for skin wounds. Improving the treatment of wound healing and tissue repair can enhance the quality of life of patients with wounds as well as reduce wound-related health care costs.

The authors would like to acknowledge with great appreciation Dr. James Futon for his valuable input and providing the A vera leaf solutions for this study. Dr. Futon unfortunately passed away during the time period of this study, and we are greatly saddened by this loss. In addition, we would like to acknowledge Stephanie J. Hustad for her technical contribution to this manuscript.

Affiliation: Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL

Correspondence: Jie Li, MD, PhD, Associate Professor, Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, RMSB 2023A, Miami, FL 33136; jli@med.miami.edu

Disclosure: This work was partially supported by the Dermatology Foundation of South Florida (Miami, FL). The funding source did not have any role during study design, data collection, analysis and interpretation of data, writing the report, or the decision to submit the article for publication.