Keywords
hypoxia - HIF-1α - VEGF-a - tooth extraction - wound healing
Introduction
After tooth extraction, immediately the empty socket is filled with blood resulting
from the hemostatic reaction in the alveolar socket, the dynamic interaction of platelets
and collagen connective tissue, as well as the balance between coagulation and fibrinolysis,
giving rise to the formation of stable blood clots embedded in the fibrin tissue.[1]
[2]
[3]
[4] The wound-healing process after tooth extraction is based on time, following the
same pattern as the wound-healing process in general with the inclusion of the socket-
and bone-healing process.[1]
A few minutes after the tooth is extracted, the blood vessels will experience vasoconstriction
as a result of platelet aggregation, causing disruption of oxygen delivery resulting
in tissue hypoxia, increased glycolysis, and a decrease in pH, which will be responded
by vasodilation; then migration of leukocytes and platelets to the wound tissue occurs.[1]
[2] Tissue hypoxia serves as a signal that stimulates many aspects of the wound-healing
process.[3]
[5]
Hypoxia is a state of reduced oxygen supply to the cellular level that is insufficient
to maintain cellular function.[6] Hypoxia can cause platelets and monocytes to release cytokines and growth factors
that affect wound healing-cells.[3]
[5]
Changes in oxygen concentration due to hypoxia will modulate cell function by stabilizing
hypoxia-induced factor-1α (HIF-1α), which is a transcription factor for many genes
that regulate adaptive responses to hypoxia.[7]
[8] Stabilization of HIF-1α as the main regulator of oxygen homeostasis and determinant
of wound-healing outcomes occurs through activation of several HIF-1α target genes.[7] HIF-1α target genes such as vascular endothelial growth factor (VEGF) are significantly
elevated in vascular smooth muscle cells that also play a role in angiogenesis, erythropoiesis,
energy and glucose metabolism to restore oxygen, nutrient delivery to the wound site,
and improving cell viability that promotes wound healing.[7]
[8]
Hypoxia can occur in an environment at an altitude with low atmospheric pressure,
where the partial oxygen pressure decreases rapidly as altitude increases, which is
known as hypobaric hypoxia (HH).[9] Hypobaric is a condition of environmental changes that occur during the rise in
altitude, including changes in air pressure, temperature, and oxygen supply. Correlations
between altitude and atmospheric pressure exist. The higher the altitude, the lower
the atmospheric pressure or the lower the partial pressure of oxygen, which becomes
a stressful condition and affects gas exchange at the cellular level. Cells unresponsive
to this stressful condition become alarmingly dysfunctional.[10] The cellular response to HH is complex and characterized by altered expression of
several genes including proteins to maintain hemostasis.[11]
Some theories explain that the accepted hypobaric hypoxic effect begins to manifest
at an altitude of 10,000 feet (3,048 m); at this height the effect of hypoxia on the
human body is clearly visible and easily recognizable.[9]
[12]
[13] HH during air travel induces several physiological reactions in the human body with
changes in gene expression, including related proteins required to maintain homeostasis.
Flying for 30 minutes resulted in decreased oxygen saturation and the expression of
10 proteins changed significantly, although the short and moderate HH of protein expression
analysis showed its relationship with immune response, protein metabolism, and hemostasis.[14]
Hypoxia does not necessarily cause damage to cells and does not necessarily have a
negative impact; rather the exposure of mild hypoxia with tolerable levels and periods
provides a protective effect (preconditioning), improving adaptive and protective
responses so that injuries from subsequent exposure to harmful stimuli are reduced.[15]
[16] Hypoxia preconditioning has better therapeutic ability than normoxic conditions,
which contributes to cell migration and cell survival, and can induce cell repair
processes.[15]
Adaptation of HH with intermittent hypobaric hypoxic efforts, as part of hypoxia preconditioning,
performed with periods of hypoxic exposure interspersed with normoxia repeatedly over
a period has several effects on tolerance to subsequent hypoxic exposure, which affects
preventing cell damage by reducing oxidative stress and inhibiting the cascade of
apoptosis.[16]
Pilots and air force flight crews routinely conduct HH training to identify hypoxic
conditions and make their bodies adapt to hypoxia. They may experience intermittent
hypobaric hypoxia (IHH) in both training and assignment.
Several studies suggest that after IHH exposure at various pressures with an interval
of 1 week, with four times of HH induction, there is an increase in HIF-1a of heart
and liver tissue after one- and two-time HH induction but continues to decrease back
to normal levels after induction of IHH three and four times, meaning that HIF-1a
is synthesized only as necessary according to the needs of liver tissue and the heart,
which then adapt to conditions of IHH.[17]
[18] To the best of our knowledge, no studies have been conducted to date to analyze
the effect of exposure to IHH on post-tooth extraction socket healing. How the post-extraction
molecular and histological changes occur, especially HIF-1α, VEGF, and angiogenesis,
in the post-extraction socket after intermittent hypobaric hypoxic exposure remains
unclear. Therefore, this study analyzed the molecular and histological changes in
HIF-1α, VEGF, and angiogenesis after IHH exposure and its effect on socket healing
after tooth extraction.
Materials and Methods
Study Design
This research is a true experimental study with a randomized post-test that only controlled
group design, using animal models of healthy adult male Sprague-Dawley rats, which
was conducted at the Integrated Research Laboratory of the Faculty of Dentistry, Padjadjaran
University, the Molecular Genetics Laboratory of the Faculty of Medicine, Padjadjaran
University, and Laboratory of the Department of Aerophysiology Lakespra dr.Saryanto
TNI Air Force.
In this study were used 45 male Sprague-Dawley rats aged between 2 and 3 months weighing
200 to 400 g, obtained from the Animal Breeding Laboratory of PT.Biomedical Technology
Indonesia, Bogor, West Java, Indonesia. One week before the hypoxia experiment, rats
were placed in a laboratory for the animal to adapt in an air-conditioned room (22 ± 3°C)
with light cycle lighting (06.00–18.00). Rats were well cared for and given mineral
water and food ad libitum every day. Prior to the start of the study, the experimental
design in this study received ethical approval from the Research Ethics Commission
of Universitas Padjadjaran, Bandung, Indonesia: 419/UN6.KEP/EC/2021.
Sample size in this study was calculated using the Federer formula as follows:
(n – 1) (k – 1) ≥ 15
where:
(n – 1) (9–1) ≥ 15
8n – 8 ≥ 15
n ≥ 2.9
The minimum sample for this study, per group, is 2.9 rounded up to 3. There was anticipation
of dropping out, so 3 becomes 5 (n = 5), meaning that the number of rats in each group is 5, so the total number of
Sprague-Dawley rats is 45 rats.
Forty-five male Sprague-Dawley rats were then randomly divided into nine groups: four
IHH groups, four normoxia groups, and one control group. They are as follows:
-
IHH groups consisting of animals were given HH exposure for 30 minutes every day in
the Hypobaric Chamber, namely group P1 (one time HH exposure, terminated on day 1,
n = 5), group P2 (3 times HH exposure, terminated on day 3, n = 5), group P3 (5 times HH exposure, terminated on day 5, n = 5), and group P4 (7 times HH exposure, terminated on day 7, n = 5).
-
Normoxia groups consisting of animals were kept under normoxia condition and placed
in the room with the same sea level, namely group K1 (terminated on day 1, n = 5), group K2 (terminated on day 3, n = 5), group K3 (terminated on day 5, n = 5), and group K4 (terminated on day 7, n = 5).
-
Control group consisting of animals was kept under normoxia condition and terminated
immediately after tooth extraction, namely group K0 (terminated on day 0, n = 5).
From all experimental animals in this study before being given treatment, the maxillary
left first molar was extracted. Experimental rats before tooth extraction were anesthetized
according to body weight (BW) and performed intraperitoneally using ketamine combined
with xylazine in the amount of 0.1 mL/10 g BW.
The dose was prepared by mixing 1.0 mL of 100 mg/mL ketamine with 0.5 mL of 20 mg/mL
xylazine. The volume of the ketamine and xylazine mixture present was added to the
saline solution until a total of 10 mL was reached. A total of 10 mL of the combination
of ketamine and xylazine was used as much as 0.1 mL/10 g BW.[19] After the anesthetic stage was reached, when the animals were in a deep sleep condition,
tooth extraction was started.
Immediately after being anesthetized, teeth of the experimental rats were extracted
using modified tooth extraction tools, namely a sterile dental periodontal surgical
instrument and arterial clamps as special pulling pliers; with unidirectional movement,
teeth were carefully pulled to avoid tooth fracture and the teeth were completely
extracted. Then post-tooth extraction socket is cleaned with sterile gauze and allowed
to undergo natural healing, and no suturing of the wound is performed on the tissue.
HH exposure in the IHH group (groups P1, P2, P3, and P4) was performed by placing
experimental rats into the Hypobaric Chamber for 30 minutes at an altitude of 18,000
feet and the air temperature was maintained at around 28°C with humidity around 58%.
HH exposure was repeated every day for intermittent hypobaric hypoxic exposure, namely
one time HH, three times HH, five times HH, and seven times HH. The HH procedure was
designed based on special training for Indonesian Air Force Soldiers.[20] The hypobaric hypoxic procedure in this experiment is shown in [Fig. 1].
Fig. 1 Altitude simulation of hypobaric hypoxia exposure procedure using the hypobaric chamber.
Hypobaric hypoxia exposure in each procedure occurred at an altitude of 18,000 feet
for 30 minutes.
The experimental rats were terminated for socket tissue sampling performed on days
0, 1, 3, 5, and 7 after tooth extraction. The IHH group was terminated immediately
at the end of each experiment after HH exposure at the ground level, the normoxic
group was terminated at the end of each experiment, and the control group was terminated
immediately on the day after tooth extraction.
The experimental animals before termination were anesthetized intraperitoneally using
a combination of ketamine and xylazine in the amount of 0.1 mL/10 g BW.[19] After the anesthetic stage was reached, and the experimental animals were deep asleep,
termination began.
Sampling from the socket tissue is performed when it is confirmed that the experimental
animal is dead. The upper jaw was cut with a scalpel, and the post-extraction socket
tissue was carefully extracted and divided into half with the thinnest separating
disk drill. Half-socket tissue per animal of approximately 20 to 30 mg was rapidly
placed in microtube centrifuge tubes, preserved with RNA later, and stored at −80°C
until use for real-time polymerase chain reaction (RT-PCR), and the remaining tissue
was fixed in formalin and processed for histological analysis.
Measurement of mRNA Expression
Measurement of HIF-1α and VEGF messenger RNA (mRNA) expression was done using one-step
RT-PCR. Samples were extracted using the RNeasy Mini Kit reagent from Qiagen. RT-PCR
was performed using the Rotor-Gene Q Software 2.3.1.49 real-time PCR system from Qiagen
with the SensiFAST SYBR No-ROX one-Step Kit for HIF-1α and VEGF. For mixing the material
into a PCR tube, use SensiFAST SYBR (2X) 10 µL composition, Primer F 0.8 µL, Primer
R 0.8 µL, Reverse Transcriptase Enzyme 0.2 µL, Ribosafe RNAse Inhibitor 0.4 µL, Nuclease
Free Water 5.8 µL, and RNA Extract 2 µL. Insert the PCR tube and then set the RT-PCR
cycle starting with the first incubation at 45°C for 10 minutes, followed by a second
incubation at 95°C for 2 minutes, then denaturation 40 cycles at 95°C for 5 seconds,
and extension at 60°C for 20 seconds. Instructions per manufacturer's protocol must
be strictly followed.
The primary sequences are as follows:
-
HIF-1α: 5′-CTTTCTCTGCGCGTGAGGAC-3′ as forward, and 5′-TTCGACGTTCGGAACTCATCCT-3′ as
reverse, and produce the amplicon size of the PCR product 149 bp.
-
VEGF-a: 5′-CTGGACCCTGGCTTTACTGC-3′ as forward, and 5′-AATTGGACGGCAATAGCTGCG-3′ as
reverse, and produce the amplicon size of the PCR product 136 bp
-
β-actin: 5′-CACCCGCGAGTACAACCTTC-3′ as forward, and 5′-CCCATACCCACCATCACACC-3′ as
reverse.
The mRNA expression was calculated using the Livak 2(−ΔΔCT) formula, by comparing
the Ct values of the treatment group with the control group.[21] Gene expression value ≥ 1 indicates that gene expression has increased compared
with control. β-actin is used as internal control. ΔCT = Ct target gene – Ct housekeeping
gene. ΔΔCT = ΔCT treatment – ΔCT control. Gene expression = 2(−ΔΔCT).
Histological Assay
Post-tooth extraction socket tissues were fixed with formalin combined with phosphate
buffer saline-formalin solution for 24 hours at 4°C. Then the samples were decalcified
with 10% ethylenediaminetetraacetic acid (EDTA) solution at pH 7.4 and stored at temperature
of 4°C for 6 to 8 weeks, depending on the degree of mineralization with EDTA, and
renewed every 3 days. Then they were trimmed and arranged into tissue cassettes and
labeled; the dehydration stage is performed next by immersing the tissues into an
alcohol solution in stages starting from 70%, then 80%, 90%, 95%, and finally 100%.
They are cleaned three times with xylene solution for 60 minutes each per cycle, then
infiltrated in liquid paraffin in three cycles by immersing the tissue into liquid
paraffin for 60 minutes each per cycle, blocking until the paraffin freezes. The tissues
were cut using a microtome slicer that were then made into slide preparations and
stained with hematoxylin and eosin (H&E); then the preparations were glued using entellan
and covered with a cover glass. The dry slides were observed under a binocular lens
microscope (Olympus Type CX31) ×400 magnification equipped with a camera with five
different fields of view. The photos produced by the camera were transferred to a
computer and evaluated with the Tool Image J software. Histological analysis of expressed
angiogenesis was quantified by counting neovascular cells with new vessel images,
then tabulated and data analyzed.
Statistical Analysis
Statistical tests analyzed the data of this research using the MegaStat V.10.4 release
3.2.4 Mac software. The data normality test used the chi-square test (p > 0.01), and the result was that all data were normally distributed. Meanwhile, the
variant homogeneity test used the Bartlett test, and the result was that all data
had a homogeneous variant. The data results are presented as mean ± standard deviation
(SD). Data were analyzed using the one-way analysis of variance (ANOVA) test followed
by post hoc t-test analysis to analyze the differences between the experimental group and the control
group. The difference was statistically significant when it showed a value of p less than 0.05. When the data results do not met the parametric test, then a nonparametric
test was performed with the Kruskal–Wallis test followed by Mann–Whitney U test analysis
to analyze differences in expression between groups.
Results
All experimental animals (n = 45) were in a safe condition and no complications occurred after tooth extraction.
The findings of each group were evaluated and calculated on days 0, 1, 3, 5, and 7
after tooth extraction. HIF-1α mRNA, VEGF mRNA, and angiogenesis expressions were
measured and compared between the experimental group (IHH and normoxia) and the control
group. HIF-1α was the main regulatory molecule for oxygen homeostasis under hypoxic
condition and determined the outcome of wound healing through the activation of several
target genes. HIF-1α expression was detected in all IHH and normoxia groups and the
control group. The results of the one-way ANOVA test found a significant difference,
p = 0.0019 (**p < 0.05), indicating that there was an effect of IHH exposure on changes in HIF-1α
mRNA expression in the socket after tooth extraction; then a post hoc t-test was performed to examine the differences between each group. Post hoc t-test results found differences in HIF-1α mRNA expression after HH exposure in the
IHH group (groups P1, P2, P3, and P4), and the normoxia group (groups K1, K2, K3,
and K4) compared with the control group (K0). The results of data analysis are shown
as mean ± SD in [Table 1].
Table 1
Differences of HIF-1α mRNA expression after IHH exposure, normoxia, and control
|
HIF-1α
|
Day 0
|
Day 1
|
Day 3
|
Day 5
|
Day 7
|
|
Control
|
1.0
|
|
|
|
|
|
IHH
|
|
3.136 ± 1.069*
|
2.509 ± 0.805[a]
|
2.073 ± 0.904
|
1.114 ± 0.560
|
|
Normoxia
|
|
2.165 ± 1.669
|
1.039 ± 0.534
|
0.751 ± 0.777
|
0.413 ± 0.300
|
Abbreviations: ANOVA, analysis of variance; HIF-1α, hypoxia-induced factor-1α; IHH,
intermittent hypobaric hypoxia; mRNA, messenger ribonucleic acid; SD, standard deviation.
Note: Values presented are mean ± SD.
a
p < 0.05, one-way ANOVA test; significantly different compared with the control group.
In the IHH group, HIF-1α mRNA expression was found after 1 time HH exposure on day
1 in the P1 group (3.136 ± 1.069) in which there was a significant increase, p = 0.0033 (**p < 0.05), compared with the control group, and after 3 times HH exposure on day 3
in the P2 group (2.509 ± 0.805), there was a decrease compared with the P1 group but
still it increased significantly, p = 0.0323 (*p < 0.05), above the control group; then after 5 times HH exposure on day 5 in the
P3 group (2.073 ± 0.904), there was a decrease approaching the control group with
a value of p = 0.1221 and, after 7 times HH exposure on day 7 in the P4 group (1.114 ± 0.560),
there was a further decrease but still above the control group with a value of p = 0.8673, indicating that the P3 and P4 groups were not significantly different from
the control group.
In the normoxia group, HIF-1α mRNA expression was found in the K1 group (2.165 ± 1.669)
on day 1, in which there was an increase with a value of p = 0.0944, and in group K2 (1.039 ± 0.534) on day 3, there was a decrease but still
it was above the control group with a value of p = 0.9542; then in the K3 group (0.751 ± 0.777) on day 5, there was a decrease below
the control with a value of p = 0.7151, and in group K4 (0.413 ± 0.300) on day 7, it increasingly decreased under
the control group with a value of p = 0.2534, indicating that the K1, K2, K3, and K4 groups were not significantly different
from the control group. The results of the analysis of differences in HIF-1α mRNA
expression are shown in [Fig. 2].
Fig. 2 Hypoxia-induced factor-1α (HIF-1α) messenger ribonucleic acid (mRNA) expression in
post-tooth extraction socket after intermittent hypobaric hypoxia exposure. (A) Group K0 (control on day 0); hypobaric hypoxic groups, namely group P1 (one time
hypobaric hypoxia [HH] exposure on day 1), group P2 (three times HH exposure on day
3), group P3 (five times HH exposure on day 5), and group P4 (seven times HH exposure
on day 7); and the normoxia groups, namely group K1 (normoxia on day 1), group K2
(normoxia on day 3), group K3 (normoxia on day 5), and group K4 (normoxia on day 7).
(B) HIF-1α mRNA expression in the socket after tooth extraction after intermittent hypobaric
hypoxia exposure in normoxia group on days 1, 3, 5, and 7. It was significantly different
compared with the control group (*p < 0.05, one-way analysis of variance test). IHH, intermittent hypobaric hypoxia.
VEGF after hypobaric hypoxic exposure was also measured in this study. VEGF as a growth
factor is a target gene for HIF-1α in a hypoxic condition, playing a role in facilitating
tissue repair by increasing vascular permeability that promotes the migration of inflammatory
cells to the wound site, stimulates angiogenesis, and grows new blood vessels that
promote wound healing. VEGF-a mRNA expression was detected in all IHH and normoxia
groups and the control group on days 0, 1, 3, 5, and 7 after tooth extraction. The
results of the one-way ANOVA test found a significant difference, p = 0.0000000064 (**p < 0.05), indicating that there was an effect of IHH exposure on changes in VEGF-a
mRNA expression in the socket after tooth extraction. Then a post hoc t-test was performed to test whether there was a difference between each group. Post
hoc t-test results found that there were differences in VEGF-a mRNA expression after exposure
to HH in the IHH group (groups P1, P2, P3, and P4) and the normoxia group (groups
K1, K2, K3, and K4) compared with the control group (group K0). The results of data
analysis are shown as mean ± SD in [Table 2].
Table 2
Differences of VEGF mRNA expression after IHH exposure, normoxia, and control
|
VEGF
|
Day 0
|
Day 1
|
Day 3
|
Day 5
|
Day 7
|
|
Control
|
1.0
|
|
|
|
|
|
IHH
|
|
1.822 ± 0.315
|
1.957 ± 0.619
|
3.547 ± 0.243[a]
|
4.153 ± 0.432[a]
|
|
Normoxia
|
|
0.712 ± 0.378
|
0.888 ± 0.539
|
1.263 ± 0.162
|
3.351 ± 0.612[a]
|
Abbreviations: ANOVA, analysis of variance; IHH, intermittent hypobaric hypoxia; mRNA,
messenger ribonucleic acid; SD, standard deviation; VEGF, vascular endothelial growth
factor.
Note: Values presented are mean ± SD.
a
p < 0.05, one-way ANOVA test; significantly different compared with the control group.
In the IHH group, VEGF-a mRNA expression was found after 1 time HH exposure on day
1 in the P1 group (1.822 ± 0.315), which increased compared with the control group
with a value of p = 0.1023, and after 3 times HH exposures on day 3 in the P2 group (1.957 ± 0.619),
there was an increase with a value of p = 0.0586, indicating that the P1 and P2 groups were not significantly different from
the control group; then after 5 times HH exposure on day 5 in the P3 group (3.547 ± 0.243),
there was a significant increase, p = 0.000008 (**p < 0.05), and after 7 times HH exposure on day 7 in the P4 group (4.153 ± 0.432),
a significant increase, p = 0.0000002 (**p < 0.05), was observed compared with the control group.
In the normoxia group, there was a change in VEGF-a mRNA expression in group K1 (0.712 ± 0.378)
on day 1 with a value of p = 0.5603 but still it was below the control group, and group K2 (0.888 ± 0.539) on
day 3 with a value of p = 0.8206 also was still below the control group, but group K3 (1.263 ± 0.162) on
day 5 began to increase above the control group with a value of p = 0.5942, indicating that groups K1, K2, and K3 were not significantly different
from the control group, while the K4 group (3.351 ± 0.612) on day 7 experienced a
significant increase, p = 0.00003 (**p < 0.05), compared with the control group. The results of the analysis of differences
in HIF-1α mRNA expression are shown in [Fig. 3.]
Fig. 3 Vascular endothelial growth factor-a (VEGF-a) messenger ribonucleic acid (mRNA) expression
in post-tooth extraction socket after intermittent hypobaric hypoxia exposure. (A) Group K0 (control on day 0); hypobaric hypoxic groups, namely group P1 (one time
hypobaric hypoxia [HH] exposure on day 1), group P2 (three times HH exposure on day
3), group P3 (five times HH exposure on day 5), and group P4 (seven times HH exposure
on day 7); and the normoxia groups, namely group K1 (normoxia on day 1), group K2
(normoxia on day 3), group K3 (normoxia on day 5), and group K4 (normoxia on day 7).
(B) VEGF-a mRNA expression in the socket after tooth extraction after intermittent hypobaric
hypoxia exposure in normoxia group on days 1, 3, 5, and 7. Significantly different
compared with the control group (*p < 0.05, one-way analysis of variance test). IHH, intermittent hypobaric hypoxia.
The amount of angiogenesis in post-tooth extraction socket after exposure to HH was
also measured on days 0, 1, 3, 5, and 7. Angiogenesis is the formation of new blood
vessels through the growth of capillary branches from existing vascular tissue, which
occurs under the activity of synergistic factor VEGF growth that plays an important
role in the wound-healing process. Angiogenesis was detected in all IHH and normoxia
experimental groups and the control group. The results of the Kruskal–Wallis test
found that there was a significant difference, p = 0.00044 (***p < 0.05), indicating that there was an effect of IHH exposure on changes in the amount
of angiogenesis in the post-tooth extraction socket; then the Mann-Whitney test was
performed to test whether there were differences between each experimental group.
The results of the Mann-Whitney test showed that there was a difference in the amount
of angiogenesis after exposure to HH in the IHH group (groups P1, P2, P3, and P4),
and the normoxic group (groups K1, K2, K3, and K4) compared with the control group
(K0).
This study found a change in the amount of angiogenesis in the IHH group. After 1
time HH exposure on day 1 in the P1 group (11.00), there was a significant increase,
p = 0.0049 (**p < 0.05), compared with the control group; then after 3 times HH exposure on day 3
in group P2 (29.80), there was a very significant increase, p = 0.000000003 (**p < 0.05), and after 5 times HH exposure on day 5 in the P3 group (32.70), it had a
significant increase, p = 0.0000000005 (**p < 0.05); then after 7 times HH exposure on day 7 in the P4 group (35.60), there was
a significant increase, p = 0.0000000001 (**p < 0.05), compared with the control group, which showed that all IHH groups (P1, P2,
P3, and P4) experienced an increase compared with the control group.
In the normoxia group, a change in the amount of angiogenesis was found. In the K1
group (9.50) on day 1, there was a very significant increase, p = 0.0217 (**p < 0.05), compared with the control group; then in the K2 group (24.60) on day 3,
there was a very significant increase, p = 0.0000000001 (**p < 0.05), and in the K3 group (26.90) on day 5 there was a very significant increase,
p = 0.00000002 (**p < 0.05); then in the K4 group (32.70) on day 7, there was a significant increase,
p = 0.0000000005 (**p < 0.05), compared with the control group, indicating that all normoxia groups (K1,
K2, K3, and K4) increased compared with the control group. The results of the analysis
of differences in the number of angiogenesis are shown in [Fig. 4.]
Fig. 4 Total angiogenesis in post-tooth extraction socket after intermittent hypobaric hypoxia
exposure. (A) Group K0 (control on day 0); hypobaric hypoxic groups, namely group P1 (one time
hypobaric hypoxia [HH] exposure on day 1), group P2 (three times HH exposure on day
3), group P3 (five times HH exposure on day 5), and group P4 (seven times HH exposure
on day 7); and the normoxia groups, namely group K1 (normoxia on day 1), group K2
(normoxia on day 3), group K3 (normoxia on day 5), and group K4 (normoxia on day 7).
(B) Total angiogenesis in the socket after tooth extraction after intermittent hypobaric
hypoxia (IHH) exposure in normoxia group on days 1, 3, 5, and 7. Significantly different
compared with the control group (*p < 0.05, Kruskal–Wallis test).
In this study, the histological results of angiogenesis were found in the post-tooth
extraction socket in the IHH group, the normoxia group, and the control group on days
0, 1, 3, 5, and 7 with H&E staining; angiogenesis had begun to appear on day 1, and
increased on day 3, then increased on day 5, and reached the highest peak on day 7
after tooth extraction. Histological picture of angiogenesis is shown in [Fig. 5].
Fig. 5 Histological test of angiogenesis in post-tooth extraction socket after intermittent
hypobaric hypoxia exposure. (A) Control group (normoxia on day 0). Intermittent hypobaric hypoxia (IHH) groups, namely:
(B) one-time hypobaric hypoxia [HH] exposure group on day 1, (C) three times HH exposure group on day 3, (D) five times HH exposure group on day 5, and (E) seven times HH exposure group on day 7. Normoxia groups, namely: (F) normoxia group on day 1, (G) normoxia group on day 3, (H) normoxia group on day 5, and (I) normoxia group day 7. Magnification ×400; yellow arrow indicates angiogenesis; hematoxylin
and eosin staining.
Discussion
In this study, we investigated the direct impact of IHH exposure on rats after tooth
extraction. After being exposed to HH in the Hypobaric Chamber, by simulating flying
for 30 minutes every day at an altitude of 18,000 feet, with exposure of 1 time HH,
3 times HH, 5 times HH, and 7 times HH, molecular changes were found in the socket
after tooth extraction in hypoxic conditions as a regulator of wound healing, describing
that flying at high altitudes will result in HH that can activate several target genes
in response to hypoxia. Early wounds will cause hypoxia due to disruption of blood
vessels that activate a series of molecular events needed in the wound-healing process.
Tissue hypoxia induces a sustained increase in HIF-1α expression as a major regulator
of oxygen homeostasis and transcriptionally regulates the expression of many other
genes that enhance the wound-healing process.[7]
The result of this study found that HIF-1α mRNA expression in the post-tooth extraction
socket increased significantly (**p < 0.05), and reached the highest after one time HH exposure on day 1, illustrating
that acute HH condition with low-level oxygen increased HIF-1α expression even higher.
HIF-1α mRNA expression began to decrease after three times HH on day 3, and further
decreased after five times HH exposure on day 5, and continued to decrease closer
to the control after seven times HH exposure on day 7. The result of this study described
that there was an increase in HIF-1α mRNA expression in the socket after tooth extraction
under acute HH condition and there was a gradual systemic adaptation at the cellular
and tissue level of socket wound after tooth extraction under intermittent hypobaric
hypoxic condition. Previous research stated that after being given IHH exposure to
experimental rat liver tissue, there was an increase in HIF-1α after one time HH and
two times HH compared with the control group; then after three times HH and four times
HH exposure, it would not increase the expression of HIF-1α, because there had been
a gradual adaptation of hepatocytes to hypoxia.[18] HIF-1α expression was significantly increased under IHH exposure condition and returned
to normal levels after repeated or intermittent hypobaric hypoxic exposure induced
a protective response causing cells to adapt to intermittent hypobaric hypoxic conditions.[16]
[17]
[18]
[22] Changes in oxygen concentration due to hypoxia would modulate cell function by stabilizing
HIF-1α, which was a transcription factor for many genes that regulated adaptive responses
to hypoxia.[7]
[23] Stabilization of HIF-1α as the main regulator of oxygen homeostasis and a determinant
of wound healing results through the activation of several HIF-1α target genes.[7]
HIF-1α mRNA expression was also found in normoxia without HH exposure on days 1, 3,
5, and 7 after tooth extraction, indicating a difference compared with the control
group. HIF-1α mRNA expression began to increase to the highest on day 1 after tooth
extraction compared with the control group, illustrating initial wound hypoxia due
to decreased oxygen levels below physiological levels, which modulated cell function
by increasing HIF-1α. In the initial wound, there was disruption of blood vessels,
which caused disruption of oxygen delivery to the wound site resulting in tissue hypoxia,
which could induce increased expression of HIF-1α.[7] HIF-1α appeared as the most active isoform during short periods (2–24 hours) of
hypoxia in some cell line.[24] HIF-1α mRNA expression on day 3 after tooth extraction began to decrease toward
the control group, then on days 5 and 7 there was a further decline until it was below
the control group, illustrating that oxygen supply during normoxic conditions returned
to normal and supplied all the cells in the wound tissue. HIF-1α expression on cells
under normoxia conditions degraded quickly and continuously, resulting in a decrease
in HIF-1α transcriptional activity.[7]
[23] In normoxic conditions, when there is sufficient amount of oxygen, HIF-1a will be
degraded, whereas under hypoxic conditions, HIF-1a undergoes stabilization by forming
heterodimers with HIF-1β and then translocating to the nucleus and binding to Hypoxia-Response
Element (HRE) elements promoting expression of target genes.[7]
[23]
[24]
[25]
In this study, VEGF-a mRNA expression in the IHH group was higher than that in the
normoxia group and the control group. The IHH group found that VEGF-a mRNA expression
increased in all groups. VEGF-a mRNA expression in the IHH group began to increase
after one time HH exposure on day 1 above the control group, then increased again
after three times HH exposure on day 3, then increased further again after five times
HH exposure on day 5, and then the highest increase occurred after seven times HH
exposure on day 7, illustrating that relatively low oxygen conditions would respond
to HIF-1α activity that could affect VEGF-a mRNA expression under HH condition that
is regulated by the HIF-1α transcription factor. The effect of IHH on VEGF expression
was highest on day 7, indicating that the formation of new blood vessels had started
to support the healing process better. VEGF could promote endothelial cell proliferation,
migration, and angiogenesis by promoting mitochondrial function.[26] Increased accumulation of VEGF via its receptor (VEGFR) on endothelial cells induced
angiogenesis and indirectly increased the oxygen supply that is required for angiogenesis.
Overall, the HIF-1α-VEGF pathway linked the process of angiogenesis.[27] Activation of HIF-1α could activate VEGF expression, and played an important role
in angiogenesis and stimulated the formation of new blood vessels at tooth extraction
sites that affected wound healing.[7]
[8] Regulation of angiogenesis by HIF-1α is very important for stimulating the formation
of new blood vessels, increasing blood supply, returning oxygen, and delivery of nutrients
to the wound site, thus increasing cell survival, which promotes wound healing.[28]
VEGF-a mRNA expression during normoxia condition without HH exposure on days 1, 3,
5, and 7 after tooth extraction in this study did not experience much change. VEGF-a
mRNA expression in the normoxia group on days 1 and 3 after tooth extraction had not
increased and was still below the control group, whereas on day 5, it began to increase,
and the highest increase occurred on day 7 after tooth extraction, which describes
the normoxic condition in line with the normal wound healing. Another study was reported
on normal wound healing, which showed that VEGF increased on days 3 to 5 and would
decrease from days 7 to 14; after tooth extraction injury, VEGF level and activity
decreased and a decrease in granulation tissue formation was observed.[29] VEGF and angiogenesis expression reached a peak in the first week after surgery,
and then levels progressively decreased until the fourth week of healing.[30] The normoxia situation was different when compared with after of IHH exposure. The
increase in VEGF-a mRNA expression occurred faster and was higher in the IHH group
compared with the normoxia group and the control group, illustrating that at the beginning
of the wound, tissue hypoxia occurred and the provision of IHH exposure with low oxygen
levels caused HIF-1α to be activated, resulting in an increase in the amount of VEGF-a
mRNA expression, which was higher than the normoxia group and the control group.
The result of histological tests in this study found that the amount of angiogenesis
in the post-tooth extraction socket after IHH exposures in the normoxia group began
to increase on days 1, 3, and 5, and reached the highest peak on day 7. The increase
in the number of angiogenesis occurred faster and higher in the IHH group compared
with the normoxia group and the control group, illustrating that at the beginning
of the wound, tissue hypoxia occurred plus the provision of IHH exposure with low
oxygen levels, activating the VEGF growth factor, resulting in an increase in the
amount of angiogenesis, which was higher than the normoxia group and the control group.
This increase in the number of angiogenesis was in line with the increase in VEGF-a
mRNA expression, illustrating that VEGF-a mRNA expression under hypobaric hypoxic
condition could be regulated by the transcription factor HIF-1α. VEGF significantly
promoted proliferation, migration, angiogenesis, and the cell cycle of endothelial
cells.[26] The relatively low O2 environment at the start of injury causes hypoxic tissue to express HIF-1α, which
activates proangiogenic transcription factors, such as VEGF, as the main regulators
of vascular growth stimulus angiogenesis.[8]
[31] Angiogenesis is the formation of new blood vessels through the growth of new capillary
branches from the existing vascular tissue and occurs under the synergistic activity
of the VEGF growth factor, which is important in accelerating tissue repair in the
wound-healing process.[28]
The limitation of this study is that this is a preliminary study to investigate the
effect of IHH exposure on changes in HIF-1α mRNA, VEGF mRNA, and angiogenesis expression
after tooth extraction in rats as the model, and this study was only performed for
7 days with seven times HH exposure and the clinical examination analysis of the post-tooth
extraction wound was not recorded.
Conclusion
In conclusion, the present study ascertained that IHH exposure increases the amount
of angiogenesis in the post-tooth extraction socket. The increased HIF-1α mRNA and
VEGF-a mRNA expressions can stimulate the formation of new blood vessels, increase
blood supply, and accelerate wound healing, thus becoming the basis for targeted and
appropriate post-tooth extraction wound-healing therapy in the future according to
the patient's adaptation to the altitude. Further research is needed to evaluate the
effect of IHH for more than 7 days with exposure to more than seven times HH and radiographic
examination.
