Keywords oral ulcer - regenerative medicine - mesenchymal stem cell - dentistry
Introduction
Accelerating the oral ulcer healing process is pivotal in managing pain and discomfort
and preventing complications.[1 ] The healing process of oral ulcers is a complex mechanism that involves various
cellular and molecular components. The ulcer healing mechanism consists of four overlapping
spatiotemporal phases: hemostasis, inflammation, proliferation, and remodeling.[2 ] Under normal conditions, the healing period for oral mucosal ulcers is between 7
and 14 days. Still, several local (e.g., local irritants, infection) and systemic
factors (e.g., diabetes mellitus, aging, vascular insufficiencies, medication) may
impede the healing process.[2 ]
[3 ] One of the essential aspects of the healing process is angiogenesis. Angiogenesis
is the mechanism of capillaries formation from pre-existing blood vessels. This biological
event is pivotal in wound healing because it facilitates adequate blood and nutrient
supply and transports many cells and molecular factors to the wound site. The previous
study has also reported that insufficient angiogenesis may lead to chronic, non-healing
wound formation. The initiation of angiogenesis requires the stimulation of several
pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) and fibroblast
growth factor-2 (FGF-2).[2 ]
[3 ]
[4 ]
The current pharmacological approaches for oral ulcers (i.e., topical steroid, non-steroid,
and herbal medication therapies) still have several clinical limitations in their
use, especially when it comes to managing chronic or extensive oral ulcer cases. For
instance, inappropriate timing and dosage of topical corticosteroid drugs can inhibit
wound healing due to their anti-inflammatory and anti-mitotic effects.[5 ]
[6 ] Karavana-Hizarcioğlu et al also identified that the healing rate of oral ulcers
treated with benzydamine hydrochloride only differed by 33% compared with the placebo
group.[7 ]
Constant scientific developments, especially in regenerative medicine, have induced
the discovery of several therapeutic biological products. Mesenchymal stem cells (MSCs)
are one of the most notorious technologies in regenerative medicine that have been widely
explored.[8 ] The recent development of MSCs is utilizing their culture medium, previously regarded
as waste, as a highly clinically promising biological product due to its content of
various bioactive factors. The collection of these factors is often referred to as
mesenchymal stem cell metabolites (MSCM).[9 ] The proteomic analysis of MSCM has reported that it contains a variety of growth
factors, cytokines, chemokines, and interventional RNA (iRNA). Bioactive molecules
contained in MSCM have been postulated to interfere with multiple biological activities
positively, especially wound healing and tissue regeneration.[9 ]
[10 ]
[11 ]
[12 ] During the wound healing process, the factors in MSCM can interact with host tissue
via the paracrine mechanism, activating several pro-regenerative pathways.[10 ] Another study reported that metabolites of mesenchymal stem cells (MSCM) are material
with good biocompatibility and can induce the proliferation of human gingival somatic
cells in in vitro studies.[13 ] These findings initiate the development of adipose mesenchymal stem cell metabolites
(AdMSCM) oral gel as the mean of MSCM application in the oral mucosa. This study will
confirm whether the application of AdMSCM oral gel can enhance the expression of VEGFA
and FGF-2, angiogenesis, and clinical outcome in oral ulcer rat models.
Materials and Methods
Study Design and Setting
Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia assigned this
study protocol ethical approval for animal laboratories with number 209/HRECC.FODM/IV/2022.
This work is a true experimental laboratory study using an analytical post-test control
group design. Lemeshow's formula determines the minimum sample; the total sample needed
is 20, with five samples for each group. Male Wistar rats (Rattus norvegicus ) weighing 250–300 g and 1 to 2 months old made up the sample. The experimental animals
were free of any oral and systemic pathologies.
AdMSCM Oral Gel Preparation
The AdMSCM oral gel was acquired from a patent owned by Research Center for Vaccine
Technology and Development, Institute of Tropical Disease, Universitas Airlangga,
Surabaya, Indonesia. The AdMSCM Gel preparation begins with the collection of plain
culture medium after the fourth passage of AdMSC culture. Then, the AdMSC culture
medium was purified using the dialysis method to remove the remnants of metabolic
products, resulting in the isolated soluble bioactive factors released by AdMSC during
culture. The purified culture media was combined with 5% hydroxypropyl-methylcellulose
(HPMC) with a 1:3 volume ratio to create AdMSCM oral gel with 30 mg/mL concentration.
Oral Ulcer (OU) Animal Model
An oral ulcer was created using an 8 g/3 mm punch biopsy tool (Premier, Plymouth,
USA) on the mucosa of the inferior incisive labial fornix. Then, the base of the tissue
was cut using a no. 15 surgical blade.[14 ] The lesion was then clinically observed 24 hours after punch-biopsy to assess the
formation of OU (clinically characterized by a white-colored lesion surrounded by
an erythematous arc). While making traumatic ulcers, experimental animals were under
general anesthesia using sodium pentobarbital which was injected intramuscularly into
the gluteal region.
The oral ulcer treatment in animals was divided into four groups, and they were treated
with 5% HPMC (control) and AdMSCM oral gel. The treatment was performed three times
daily, using disposable micro applicators (Cotisen, China), for 3 and 7 days. The
termination of experimental animals was performed using the cervical dislocation technique.
Before termination, the animals were anaesthetized using a single intraperitoneal
injection of pentobarbital 50 mg/kg (pentobarbital solution, no. cat: P-010, Sigma
Aldrich) systemically (20–40 mL).
Clinical Evaluation of Ulcer's Diameter
The major ulcer diameters of each sample were measured using a fine precision ruler
(Fisherbrand, Pittsburg, USA) with a 1 mm increment. The measurement was conducted
while the animals were under general anesthesia.
Tissue Preparation
An excisional biopsy of the inferior fornix labial mucosa was conducted for tissue
harvesting. The tissues were then fixated in a 10% neutral buffer formalin (NBF) solution
(Sigma Aldrich, California, USA) for three days. After that, the tissues were washed
three more times with PBS (OneMed, Sidoarjo, Indonesia) for 5 to 10 minutes each.
Then, the tissues were embedded in paraffin and sectioned with a rotary microtome
to obtain HPA slides.
Histological Evaluation of Angiogenesis
The histopathology slides were processed and stained with hematoxylin–eosin (HE).
Then, the angiogenesis of each slide was observed using a light microscope with 100 × ,
400 × , and 1000× magnification (Nikon, H600L, Japan) at the Dental Research Center,
Faculty of Dental Medicine, Universitas Airlangga. The calculations of the angiogenesis
number were performed in five different fields of view by two observers.
Immunohistochemistry
The immunohistochemistry staining was used to evaluate VEGFA and FGF-2 positive expressions
in the tissues. The histopathology slides were processed immunohistochemically using
horse radish-labeled monoclonal antibodies (anti-VEGFA #SC-7269 (Santa Cruz Biotechnology Inc., California, USA), FGF-2 #SC-74412 (Santa Cruz Biotechnology Inc., California, USA)), and 3–3′ diaminobenzidine (DAB) (Abcam, USA). Then, the
positive expression of the protein marked by brown precipitate on the cells of the
oral ulcer site was observed using an inverted light microscope with 100 × , 400 × ,
and 1000× magnification in five different fields of view by two observers.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0 ( for MacBook, v9.4.1, San Diego, USA). The normality test was performed using the Shapiro–Wilk
test and the homogeneity test using Levene's test. If the data obtained met the requirements
for parametric tests, a one-way analysis of variance (ANOVA) test (p < 0.05) is going to be conducted to find the differences between all groups. If the
data did not meet the requirements for a parametric test, a nonparametric test was
performed using the Kruskal–Wallis test (p < 0.05). Then, the multiple comparison post-hoc test using Tukey's HSD was performed
to uncover specific differences between groups.
Results
In this investigation, it was discovered that the data on the ulcer diameter, angiogenesis,
and the expression of VEGFA and FGF-2 was homogeneous and fitted with Gaussian distribution
(p > 0.05); thus, comparative parametric analysis using one-way ANOVA was performed
with statistical significance is assumed at the p < 0.05.
Clinical Outcome
Clinical images showing the oral ulcers after treatment for 3 and 7 days can be seen
in [Fig. 1A-1D ]. The AdMSCM oral gel treatment groups reveals substantial evidence of a significant
reduction in ulcer diameter found after treatment for three (p = 0.0002) and seven days (p < 0.0001) ([Fig. 1E ]; [Table 1 ]).
Table 1
Ulcer diameter, angiogenesis, and the expression of FGF-2 and VEGFA in an oral ulcer
rat model
Treatment Duration
Group
Ulcer Diameter
Angiogenesis
VEGFA
FGF-2
Mean ± SD
Three days
control
8.4 ± 1.140
4.8 ± 1.304
2.2 ± 0.837
2.2 ± 0.837
AdMSCM oral gel
4.6 ± 1.140
4.2 ± 1.304
4.0 ± 1.000
4.8 ± 1.304
Seven days
control
4.8 ± 0.837
5.2 ± 0.837
7.0 ± 1.581
7.0 ± 1.581
AdMSCM oral gel
0.8 ± 0.837
9.0 ± 1.581
10.0 ± 1.581
11.40 ± 1.140
p -Value
0.0001*
0.0001*
0.0001*
0.0001[* ]
Abbreviations: AdMSCM, adipose mesenchymal stem cell metabolite; FGF-2, fibroblast
growth factor-2; OU, oral ulcer; SD, standard deviation. VEGFA, vascular endothelial
growth factor A.
* Significant difference of one-way ANOVA represented by p < 0.05.
Fig. 1 The clinical of oral ulcer in animals. (A ) control after three days of treatment; (B ) AdMSCS oral gel after three days of treatment; (C ) control after seven days of treatment; (D ) AdMSCS oral gel after seven days of treatment and (E ) The AdMSCS oral gel treatment significantly increased the oral ulcer healing. ***p < 0.001; ****p < 0.0000
Angiogenesis
The histologic assessment of angiogenesis in the oral ulcer site is represented in
[Fig. 2A-2D ]. The AdMSCM oral gel treatment on oral ulcers for three days did not significantly
increase the angiogenesis (p = 0.9597). However, significantly higher angiogenesis was found seven days after
AdMSCM oral gel treatment (p = 0.0001; [Fig. 2E ]; [Table 1 ]).
Fig. 2 The histopathology of oral ulcer with hematoxyline eosin staining in control and
AdMSCM oral gel treatment for three days (A and B ) and seven days (C and D ). The AdMSCM oral gel treatment increased the angiogenesis (E ). ** p < 0.01; ***p < 0.001; ns: not significant
FGF-2 Expression
The histopathology view of the FGF-2 expression pattern in the oral ulcer site can
be seen in [Fig. 3A-3D ]. Brown precipitates in the epithelial layer and connective tissue of the ulcer site
mark FGF-2 expression. According to the results of the one-way ANOVA, there were statistically
significant differences between all groups (p = 0.0001; [Table 1 ]). The AdMSCS oral gel demonstrates a significantly higher FGF-2 expression after
three (p < 0.0001) and seven days (p < 0.0001) compared with the control ([Fig. 3E ]).
Fig. 3 The histopathology of oral ulcer with immunohistochemistry staining in control and
AdMSCM oral gel treatment for three days (A and B ) and seven days (C and D ). The AdMSCM oral gel treatment increased the FGF-2 expression (E ). *p < 0.05; ***p < 0.001; ****p < 0.0000
VEGFA Expression
The VEGFA expression pattern is marked by brown precipitates in the epithelial layer
and connective tissue of the ulcer site, as seen in Figure 4A-4D. The VEGFA expression
after AdMSCM oral gel treatment for 3 and 7 days was higher than the control (p = 0.0001 and p < 0.0001, respectively; [Fig. 4E ]).
Fig. 4 The histopathology of oral ulcer with immunohistochemistry staining in control and
AdMSCM oral gel treatment for three days (A and B ) and seven days (C and D ). The AdMSCM oral gel treatment increased the VEGFA expression after seven days of
treatment (E ). *p < 0.05; ***p < 0.001; ****p < 0.0000
Discussion
AdMSCM oral gel contains a collection of bioactive molecules, such as growth factors,
cytokines, chemokines, and noncoding RNAs (e.g., micro RNA [miRNA]) produced by MSCs
during culture. The study by Park et al reported that AdMSCM contains more than 40
types of growth factors, mainly EGF, FGF-2, IGF-1, and HGF, which are reported to
be able to accelerate the wound healing process via a paracrine mechanism.[9 ]
[10 ] The same study also reported that the administration of AdMSCM was able to increase
the activity of the PI3K/Akt or FAK/ERK1/2 pathways that play a role in regulating
the proliferation and migration activity of various skin and oral mucosal cells, such
as fibroblasts, keratinocytes, and vascular endothelial cells that will induce the
process of wound contraction.[10 ] In addition, another study by Nugraha et al reported that MSCM is biocompatible,
nontoxic, and able to increase the proliferation of human somatic cell culture 12 hours
after application.[13 ] These studies supported the finding of this study that reported the excellent clinical
outcome of the AdMSCM oral gel treatment groups.
Limiting inflammatory activity in wound healing is essential for creating an optimal
healing process.[15 ]
[16 ] Previous studies reported that a prolonged inflammatory phase of wound healing would
cause the proliferative phase not to be achieved, which may lead to the formation
of chronic wounds.[2 ]
[5 ] In addition to proregenerative effects, AdMSCM oral gel can exert immunomodulatory
effects produced by various anti-inflammatory components.[11 ] AdMSCM contained various soluble anti-inflammatory cytokines (such as TGF-β, IL-10,
and PGE-2), extracellular proteins that act as a resolution-associated molecular pattern
(RAMPs) (e.g., IDO, HSP10, HSP70), and interference RNAs (e.g., the miR-family).[17 ]
[18 ]
[19 ] According to research by Zriek et al, the immunomodulatory components of MSCM can
interact with immune cells to activate anti-inflammatory regulatory phenotypes.[20 ]
Macrophages with the M1 phenotype are one of the primary mediators of inflammation
in wound healing. Various anti-inflammatory cytokines (e.g., TGF-β and IL-10) present
in the AdMSCM oral gel can interact with their respective receptors on macrophages
and trigger the polarization of pro-inflammatory macrophages (M1) to anti-inflammatory
macrophages (M2). In addition, anti-inflammatory cytokines can also induce the activation
of M0 macrophages through the alternative pathways. This theory is proven by Holthauss
et al where it was reported that the administration of preconditioned AdMSCM to M0
cells was able to increase the expression of Arg1 and MerTK genes, which are genes
that are highly expressed in the activation of the alternative pathway of macrophages
(M0 to M2 polarization).[18 ] The role of RAMPs in the resolution of inflammation was also demonstrated in the
study by Borges et al, where HSP70 can produce an anti-inflammatory effect through
the activation of the TLR-2 pathway, which will trigger the activation of downstream
ERK proteins to induce the production of IL-10.[21 ] Recent studies have also demonstrated the critical role of miRNAs in the resolution
of inflammation in physiological wound healing.[22 ]
[23 ] The immunomodulatory effect of miRNA is evidenced by the reported role of miR-let7b
in triggering M1 to M2 polarization.[24 ] Accelerated resolution of inflammation in the wound healing process can accelerate
the transition to the proliferative phase so that it will indirectly accelerate wound
closure, as illustrated in the results of this study.[23 ] In addition, miRNAs have also been reported to regulate the proliferative phase
of wound healing, specifically at the stages of re-epithelialization, angiogenesis,
and granulation tissue formation.[22 ]
This study also reported that the level of angiogenesis that occurred in the treatment
group was significantly higher than in the control group (p < 0.05). Angiogenesis is the growth of new capillaries from existing blood vessels.[4 ] This event occurs in the proliferative phase of the wound-healing process.[2 ] This process is crucial to creating an effective and optimal wound-healing process.[4 ] The process of angiogenesis is initiated by an injury that causes microvascular
endothelial cells (MEC) that line the tunica intima of blood vessels to be activated
by hypoxia and pro-angiogenic factors such as VEGF and FGF-2.[25 ] Angiogenesis primary function in wound healing is to facilitate oxygen and nutrient
supply and transport cells and molecules. The content of pro-angiogenic factors (e.g.,
VEGF and FGF-2) in the AdMSCM oral gel is thought to have contributed to the findings
in this study. Previous study reported similar results, where the application of AdMSCM
could significantly increase the expression of CD-31 (platelet endothelial cluster
adhesion molecule-1/PECAM1), a specific marker molecule of vascular endothelial cells.[10 ] Angiogenesis can also be induced by miRNAs contained in MSCM.[19 ] Previous studies reported that MSCM contains several pro-angiogenic miRNAs, such
as miRNA-23a.[19 ] miRNA-23a can inhibit the prolyl hydroxylase ½ (PHD ½) gene, leading to the accumulation
of HIF-1α in vascular endothelial cells, thereby inducing angiogenesis.[26 ] In addition, miRNA-23a can increase vascular permeability and cell migration which
is crucial in angiogenesis.[22 ]
As the primary pro-angiogenic factor, observing VEGFA expression may indicate the
potency of AdMSCM oral gel in enhancing angiogenesis.[4 ] This study also found that the expression of VEGFA in the AdMSCM oral gel treatment
group on the third and seventh days was significantly higher than in the control group
(p < 0.05). A similar result was also reported by Sunarto et al, where the administration
of MSCM can increase VEGF levels in skin wound model.[27 ] In addition, similar results were also found in the observation of FGF-2 expression,
where the mean FGF-2 expression in the AdMSCM oral gel treatment group on the third
and seventh days was significantly higher than the control group (p < 0.05). The importance of FGF-2 in wound healing is not only limited to their function
to induce angiogenesis, but also the proliferation of fibroblast in granulation tissue
formation phase. FGF-2 able to induce proliferation of human dermal fibroblast via
the activation of ERK ½ and JNK pathways.[28 ]
[29 ] The findings that the expression of these two growth factors in the treatment group
was significantly higher than in the control group can be explained by the significant
role of M2 macrophages in producing various growth factors that can induce wound healing.[2 ]
[30 ] Larjava et al reported that M2 macrophages could produce various growth factors
relevant to the wound healing process, such as VEGFA, IGF-1, FGF-2, and HGF.[2 ]
[31 ] As previously mentioned, the immunomodulatory content of the AdMSCM oral gel was
able to induce pro-reparative M1 to M2 polarization of macrophages.[30 ]
[32 ]
Conclusion
This investigation reveals that the topical application of AdMSCM oral gel is able
to enhance the clinical outcome, angiogenesis, and expression of VEGFA and FGF-2 in
the oral ulcer animal model (R. novergicus ). Further exploration through other approaches is still required to evaluate AdMSCM
oral gel potency in inducing oral ulcer healing.
