The Effects of Aloe vera on Wound Healing in Cell

Proliferation, Migration, and Viability

Authors
Eric Teplicki
 
Qianli Ma
 
David E. Castillo
 
Mina Zarei
 
Adam P. Hustad
 
Juan Chen
 
Jie Li
Keywords
Aloe vera
 
wound healing
 
cell migration
 
cell proliferation
 
cell viability

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September 2018
Issue: Volume 30 - Issue 9 - September 2018
ISSN: 1044-7946
Index: Wounds 2018;30(9):263–268.

Abstract
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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Introduction
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 models11-16; 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.20 Aloe 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 studies18,21 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.
Materials and Methods
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.
Results
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).
Discussion
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 results17,25 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.
Limitations
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.
Conclusions
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.
Acknowledgments
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.