The effect of Aloe vera (L.

) on the expression of wound healing factors

(TGF β1) in oral mucosa Rattus norvegicus: An in- vivo study
Abstract
Aloe vera (L.) in several cultures has been used as for herbal medicines It, and is
also known as the healing plant. This study will analyze the effects of Aloe vera
(L.) on the healing process of wounds in oral mucosa of twenty-four male Rattus
norvegicus. Wound length, expression of TGF �1 and inflammation effect were
analyzed by immunohistochemistry and hematoxylin-eosin(HE) staining. Crude
extracts of Aloe vera (L.) were topically applied topically. A signficant wound
length reduction (p<0.005) was observed at the third (p=0.001) and sixth day
(p=0.003). TGF β1 expression was significantly increased at the third (p=0.041)
and sixth day (p=0.015). There areis no significant reduction of inflammatory
cells either on the third (p=0.699) or sixth day (p=1.0). The conclusion of this
study showeds Aloe vera (L.) enhanced oral mucosa wound healing, both
clinically and based ony TGF β1 marker. There areis no significant reduction of
inflammatory cells.
Keywords: Aloe vera, herbal medicine, oral mucosa wound healing, wound
length, TGF β1, inflammatory cells.
Introduction
Tooth extraction is one of the treatments that may cause injury. To restore the integrity
of damaged tissue, effective wound healing requires the supply of materials and
nutrients (Subramanian et al. 2006; Moghbel et al. 2007). Medicinal plants usually
contain materials and nutrients necessary to accelerate the process of wound healing.
Aloe vera (L.) in several cultures has been widely used as herbal medicines. This plant
has a famous remarkable effect in wound healing. The crude extract of Aloe vera (L.)
contains various components, generally classified into saccharides (polysaccharides,
monosaccharides and glycosides), quinones (anthraquinones), minerals, proteins
(bradykinase), lipids, and supplementary substances (Surjushe et al. 2008; Gupta &
Malhotra 2012; Rahman et al. 2017). Many researchs proved that the use of Aloe vera
(L.) gel extracts resulted in faster wound healing (Radha & Laxmipriya 2015; Hashemi
et al. 2015; Akaberi et al. 2016). This extract was reported able to increase wound
contraction and collagen synthesis, stimulate the macrophages and proliferation of
fibroblasts, and have anti-inflammatory effects (Radha & Laxmipriya 2015; Hashemi et
al. 2015).
Studies on skin wound healing in rats and rabbits had reported that saccharides
and anthraquinones are present in Aloe vera (L.). Saccharides can accelerate the healing
process which involves skin contraction and collagen synthesis. Studies also showed
high expression of Transforming growth factor �1 (TGF �1) gene (Atiba et al. 2011;
Hashemi et al. 2015). TGF �1 is a multifunctional growth factor, that stimulates
proliferation of fibroblasts, differentiation of myofibroblasts, and enhances the
formation of extracellular matrix (ECM)(Li & Flavell 2008; Atiba et al. 2011;
Moustakas & Heldin 2016). Immediately after skin injury, TGF �1 will leave from skin
wound by inducing platelets degranulation. TGF �1 shows increased mitotic index in
the study of human’s skin fibroblasts cells (Hashemi et al. 2015). Besides saccharides,
other important molecules in Aloe vera (L.) are anthraquinones. It have played an
important role in inflammation by inhibiting the cyclooxygenase pathway and reduces
prostaglandin E2 production (Hashemi et al. 2015; Zhou et al. 2015).
In contrast, the healing of oral mucosa is slightly different from the skin.
Fibroblasts in oral mucosa phenotypically resemble fetal fibroblasts (Nanci 2012). This
study will analyze the effects of Aloe vera (L.) extract on the healing process of oral
mucosa wound, based on the wound length, the expression of TGF �1, and its effect on
inflammation.
Material and Methods
Experimental animals
This study was approved by the animal care and experimentation committee, Faculty of
Medicine, Maranatha Christian University – Immanuel Hospital Bandung. An effort was
made to minimalize the number of animals used in this study. All the rats were housed
at Animal Facility of Pharmacology Therapy and Clinic Laboratory, Faculty of
Medicine, Universitas Padjadjaran Bandung, with food and water ad- libitum under
controlled temperature (24 ± 3˚ C), 12-hours light, 12-hours dark cycle (light on 06.00
am until 06.00 pm). Twenty-four male wistar rats (Rattus norvegicus) weighing 200-250
grams by the age of 40-60 days were purchased from Faculty of Veterinary Medicine,
Bogor Agricultural University. The rats were divided into four groups (n=6/group).
Groups one and two were given injuries without treatment of topical Aloe vera (L.).
Groups of three and four were given injuries and Aloe vera (L.) treatment. All rats went
under anesthesia with ketamine (10 mg/kg body weight) given through intramuscular
injection.intramuscularly, and were cut along 3mm on the mandibular labial gingiva. At
the end of the experimental period, three days for group one and three and six days for
group two and four, the rats were terminated by anesthesia and cervical dislocation.
Aloe vera (L.) Extract
The extract of Aloe vera (L.) have been tested and standardized at the Faculty of
Pharmacy Universitas Padjadjaran. This extract was topically applied twice daily. On
the third day, mucosa wound length measurement, and immunohistochemically were
analyzed by specific antibody and hematoxylin-eosin staining were carried on groups 1
and 3. The same procedure was also conducted at sixth day for groups 2 and 4.
Planimetric measurement of mucosa wound length
The changes in the length of the mucosa wound contraction were monitored
planimetrically by tracing the mucosa wound margin with calipers every alternate day
without causing any damage to the wound area. The measurement of mucosa wound
length was expressed as the mucosa wound contraction.
IHC of TGF β1
Immunohistochemically (IHC) staining of the mucosal tissue sections was performed
using anti-TGF β1 with labeled streptavidin-biotin method (LSAB) that uses the
primary TGF β1 (R & D systems, Minneapolis, MN, USA), with the instructions of the
LSAB-HRP System kit. Unstained tissue sections were dewaxed and rehydrated in
xylene and graded ethanol. The optimal protocols for TGF-β1 immunohistochemistry
were determined by testing primary antibody dilutions and staining conditions, which
was conducted at the Anatomic Pathology Laboratory, Hasan Sadikin General Hospital
Bandung.
The staining for TGF-β1 on mucosal tissue was evaluated by a pathologist who
was blinded to the experimental data. The immunoexpressing level is determined by the
distribution of cells that showed immunoreactions and immunohistochemically color
intensity using a light microscope (Olympus CX21FS1, Tokyo, Japan) with a 400x
magnification. The immunoreacted cells are described as brown,round-shaped cells.
Distribution of cells with positive immunoexpressionng was counted semi-
quantitatively by a scoring of 0 to 3 (Fedchenko & Reifenrath 2014); 1 (focal) if there
were <20% of immunoreacted cells; 2 (heterogeneous) if there were 20-50% of
immunoreacted cells; 3 (diffuse) if there were >50% immunoreacted cells.
The intensity of immunohistochemical staining can be assessed by a scale of 0 to
3 (Fedchenko & Reifenrath 2014); 0 (negative) if there is no brown granular staining; 1
(weak) very smooth, light brown granular staining; 2 (moderate) more brown granular
staining; 3 (strong) dark brown granular staining.
HE staining
Mucosal tissue sections (4–5 μm) were dewaxed and dehydrated using ethanol solutions
and stained with hematoxylin-eosin (HE), as the standard procedure in this condition.
All sections were observed and the pictures were photographed with a light microscope.
The representative fields within each section were randomly chosen and captured under
a 100x magnification. The integrated optical density (IOD) in each image was measured
with the same setting for all the slides, and the density was calculated as IOD/total area
of each image.
HE staining was used to examine the effect of Aloe vera (L.) on inflammatory
reactions during mucosa wound healing. The percentage of present inflammatory cells
were scored with a scale of 0 to 3 (Erben et al. 2014); 0 (negative) if no inflammatory
cells were found; 1 if there are <20% inflammatory cells; 2 if there are 20-50%
inflammatory cells; 3 if there are > 50% inflammatory cells.
Data analysis
Statistical comparison was performed using T-test and Mann-Whitney. The p value of
less than 0.05 (p<0.05) was considered significant.
Results
Wound length
In Aloe vera (L.) treated groups (3 and 4), the length of mucosa wound significantly
showed more reduction compared to the untreated group (p = 0.001 for 3 rd-day; p =
0.003 for 6th-day). On the third day, it rangedis ranging from 1.45 mm (group 3) and
2.36 mm (group 1) and reachedbecame 0.037 mm (group 4) and 1.56 mm (group 2) on
the sixth day. The representative data can be seen in the table 1. [t]Table 1 near here [/t].
Table 1. Length of the mucosa wound on the third and sixth day
Length of the mucosa wound (mm)
Mean ± SD p-value
Without Aloe vera (L.) Aloe vera (L.)
 (n=6/group) (n=6/group)
Third day group 1 2.36 ± 0.37 group 3 1.45 ± 0.095 p=0.001
group 2 1.56 ± 0.32 group 4 0.037 ±
Sixth day p=0.003
0.057
Note: The length of the labial mucosa wound from each group were analyzed using Mann-
Whitney test. It significantly decreases in the third day groups (1 and 3) (p=0.001, p<0.05)
and six day groups (2 and 4) (p=0.003, p<0.05). Topical application of Aloe vera (L.)
showed a significantly decrease of mucosa labial wound length.
Immunoexpressionng level of TGF β1
Immunoexpressionng level of TGF β1, from groups (1 and 2) without Aloe vera (L.)
shows an average weak intensity (1) and distribution, either on the third or sixth day. In
groups (3 and 4) with Aloe vera (L.), a strong intensity and distribution was observed
(3). This is shown in figure 1. [f]Figure 1 near here [/f].
Figure 1. TGF β1 expression observed using a light microscope with 400x
magnification. (a) Immunoreacted cells distribution (arrow) <20% (1: focal) represent
the untreated group (not received topical application of extract Aloe vera (L.)). (b)
Immunoreacted cells distribution (arrow) > 50% (3: diffuse) represent the treated group
(received topical application of Aloe vera (L.)). (c) Weak intensity (category 1) of
brown granular staining represent the untreated group. (d) Strong intensity (category 3)
of brown granular staining represent the treated group.
The intensity and distribution of TGF β1 expression lead to a significant increase
in the third (p = 0.041) and sixth day (p = 0.015). The comparison can be seen in the
table 2. [t]Table 2 near here [/t].
Table 2. Intensity and distribution of TGF β1 expression immunoreacted cells on the
third and sixth day
Treatment
without Aloe vera Aloe vera (L.)
TGF β1 Expression p-value
(L.) (n=6/group)
(n=6/group)
Third Intensity 1 (weak) 4 0
day 2 (medium) 2 3
3 (strong) 0 3
Group 1 Group 3 0.041
Distribution 1 (focal) 4 2
2 (heterogenous) 1 4
3 (diffuse) 1 0
Sixth Intensity 1 (weak) Group 2 4 Group 4 0 0.015
day 2 (medium) 1 1
3 (strong) 1 5
 Distribution 1 (focal) 5 2
2 (heterogenous) 1 4
3 (diffuse) 0 0
Note: Categorization of IHC staining intensity by a scale of 0 to 3; 0 (negative) if there is no
brown granular staining; 1 (weak) very smooth, light brown granular staining; 2
(moderate) more brown granular staining; 3 (strong) dark brown granular staining.
Categorization of immunoreacted cells distribution by a scale of scoring 0 to 3; 0
(negative) if no immunoreacted cells were found; 1 (focal) if there were <20% of
immunoreacted cells; 2 (heterogeneous) if there were 20-50% of immunoreacted cells; 3
(diffuse) if there were >50% immunoreacted cells. In this experiment, no group were
categorized as 0. TGF β1 expression led to a significant (p<0.05) increase in the value of p
= 0.041 on the third day and p = 0.015 on the sixth day, which is confirmed by Mann-
Whitney test.
Number of inflammatory cells with HE staining
There is no significant decrease in number of inflammatory cells between both groups :
treated and not treated with Aloe vera (L.) as showed by Mann-Whitney test results (p =
0.699 for 3rd-day; p = 1.0 for 6th-day). The comparison can be seen in the table 3.
[t]Table 3 near here [/t].
Table 3. Percentage of inflammatory cells on the third and sixth days in both group of
treatments
Treatment
Inflammatory cells (%) without Aloe vera Aloe vera p value
(n=6/group) (n=6/group)
Third day 1 (<20%) 3 4
2 (20-50%) 2 1
3 (>50%) Group 1 1 Group 3 1 0.699

Sixth day 1 (<20%) 3 3

2 (20-50%) Group 2 1 Group 4 1 1.0
3 (>50%) 2 2
Note: Categorization off inflammatory cells were counted semi-quantitatively by a scale
of 0 to 3; 0 (negative) if no inflammatory cells were found; 1 if there is <20% of
inflammatory cells; 2 if there is 20-50% cell inflammation; 3 if there is > 50% of
inflammatory cells. In this experiment, no group were categorized as 0. Inflammatory
cells after analyzed using Mann-Whitney test on the third day have a p-value of = 0.699
and p-value = 1.0 on the sixth day. No significant (p>0.05) decrease was observed,
either on the third or sixth day.
 HE staining showed the number of inflammatory cells in the mucosa labial
wound healing process under light microscope. Semi quantitatively analyses were done
based on the percentage of inflammatory cells, and can be observed are present in figure
2. [f]Figure 2 near here [/f].
Figure 2. HE staining for the number of inflammatory cells (numerous round-shaped
cells) in the mucosa wound healing process under a light microscope with 100x
magnification. (a) Number of inflammatory cells (arrow) <20% (category 1). (b)
Number of inflammatory cells (arrow) > 50% (category 3).
Discussion
Aloe vera (L.), or its botanical name Aloe barbadensis Miller, is a part of the Liliaceae
family. This plant has thick leaves to survive dry climate. The mucilaginous layer in the
inner leaves are thought to be responsible for the majority of the plant’s therapeutic
properties. This layer gel contains approximately 72% saccharides (polysaccharides,
monosaccharides and glycosides), 16% minerals, 7% proteins (bradykinase), 4% lipids,
and 1% supplementary substance. The upper yellow sap contains phenolic compounds,
such as anthraquinones. The therapeutic properties of Aloe vera (L.) has been described
as having wound healing, anti-inflammatory properties, radiation damage repair,
antibacterial, antiviral, antifungal, antidiabetic, antineoplastic, hematopoietic
stimulation, and antioxidant (Surjushe et al. 2008; Rahman et al. 2017).
Wound healing properties of Aloe vera (L.) on the skin hads been previously
proven by many researchers. This research proved its existence in oral mucosa wounds.
Topical application of Aloe vera (L.) extract on oral mucosa showed significant effect
on rats and rabbits skin (Surjushe et al. 2008; Atiba et al. 2011; Hashemi et al. 2015).
The length of the wound reduced due to increased skin contraction at the wound point,
collagen synthesis, and fibroblast proliferation (Atiba et al. 2011; Hashemi et al. 2015).
The polysaccharides are responsible for the majority of the biological activities of this
plant (Jettanacheawchankit et al. 2009; Rahman et al. 2017). The polysaccharides
consist of linear chains of glucose and mannose molecules, in which acemannan and
glucomannan are the forms that have the most important functions (Rahman et al.
2017). This molecule plays an important role in several phases of wound healing
(Jettanacheawchankit et al. 2009; Gupta & Malhotra 2012). Three phases of wound
healing are thrombosis and inflammation, proliferation and formation of new tissue, and
remodeling or maturation phase (Hashemi et al. 2015).
One of the most important immune cells involved in the inflammation phase
until the last phase of wound healing are macrophages (Delavary et al. 2011).
Macrophage levels start to increase during the phase of inflammation, with the peak
during the formation of granulation tissue and decline in the maturation phase.
Previous in vitro studies reported that mannose molecules bind to mannose
transmembrane receptor on the surface of macrophages and induce the intracellular
signaling pathway (Jettanacheawchankit et al. 2009; Atiba et al. 2011; Delavary et al.
2011). These receptors contain an N-terminal cysteine-rich domain, a fibronectin type II
domain, multiple C-type lectin-like domains (CTLDs), and a C-terminal cytoplasmic
domain. The CTLDs recognize polysaccharide chains ending with mannose, fucose, or
N-acetylglucosamine (Jettanacheawchankit et al. 2009). To confirm this interaction,
future studies are still required.
Macrophages, platelets, injured cells, fibroblasts, and other cells involved in
wound healing coordinate with each other through cellular signaling by the help of
various types of growth factors. Growth factors are proteins with heavy molecular
weight, which secretes and start autocrine and paracrine signalling in various cellular
processes. Growth factor that has a substantial role is TGF � (Atiba et al. 2011;
Hashemi et al. 2015). The major source that is secreted by this growth factor are
macrophages. TGF � has three isoforms, TGF β1, β2, and β3 (Delavary et al. 2011).
Between these three isoforms, high expressions of TGF β1 are associated with scarless
conditions and enhanced the wound contraction rate and ECM production in vivo. The
expression level of TGF β1 reflects the distribution of leukocytes or/and macrophages.
In this study we observed an immunoreaction against anti-TGF β1 antibodies (Fig. 1a-
b). TGF β1 plays a role in chemotaxis, fibroblast proliferation, and collagen metabolism
(Hashemi et al. 2015). These molecules will interact with growth factor receptors on the
fibroblast, thereby stimulating its activity and proliferation. The fibroblast is one of the
cells that plays an important role in the proliferative phase of wound healing, and
fibroblasts are needed to maintain skin integrity (Jettanacheawchankit et al. 2009; Nanci
2012).
 In the early phase, Aloe vera (L.) serves as a dressing that keeps the wound
moist (Rahman et al. 2017). It was reported that Aloe vera (L.) increases collagen
synthesis, changes collagen composition, enhances tensile strength and causes collagen
cross-linking to damaged tissues (Arijani & Khoswanto 2008; Surjushe et al. 2008). The
mechanism of Aloe vera (L.) in increasing collagen synthesis still requires further study,
as well as the effects of Aloe vera (L.) on growth factors.
Anthraquinones, the phenolic compounds in Aloe vera (L.) leaves, has strong
anti-inflammatory effects (Rahman et al. 2017). This effect is possible by inhibiting the
cyclooxygenase pathway and reducing prostaglandin E2. The other molecules that have
anti-inflammatory effects are C-glycosyl chromone extracts (glycosides) and
bradykinase (peptidase). Bradykinase is shown to break down bradykinin, an
inflammatory substance that induces pain (Gupta & Malhotra 2012). In this study, the
anti-inflammatory effect of Aloe vera (L.) was observed by detecting the presence of
inflammatory cells (fig. 2a-b). The percentage of inflammatory cells did not show a
significant reduction (table 3). This may be caused due the examination being held on
the third and sixth day which is still in the inflammatory phase. The most prominent
inflammatory cell within three to five days is macrophages. Reduction of macrophages
during these phases will cause delay of wound healing in mouse and rabbit models
(Subramanian et al. 2006; Atiba et al. 2011; Delavary et al. 2011). More studies are still
required to prove the anti-inflammatory effect of this plant, by increasing the treatment
time and modification of existing research methods.
Conclusions
Aloe vera (L.) enhances oral mucosa wound healing, significantly reduces wound
length, increases TGF β1 expression but causes no significant reduction of
inflammatory cells.
Acknowledgements
We would like to thank Hj. Bethy Suryawathy Hernowo, M.D., Sp.PA(K)., Ph.D. for the
pathological analysis. This work is supported by Maranatha Christian University
Scholarship, and we would like to thank Universitas Padjadjaran for supporting our
publication fundings.
Competing Interests
The authors declared no competing interests.