Keywords diode laser 650-nm irradiation - biomodulator - odontoblast like cells - VEGF-A -
TGF-β1
Introduction
Recent studies about diode laser for vital pulp therapy are now developing. Main goal
of this therapy is to initiate formation of reparative dentin. Vital pulp therapy
can be done by application of some materials, such as calcium hydroxide, mineral trioxide
aggregate (MTA), and biodentine, or even by another strategy such as laser irradiation,
ozone technology, silver diamine fluoride, or others.[1 ]
[2 ] Diode laser 810 nm has more significant hemostatic effect and antibacterial advantage
compared with chemical agents (ferric sulfate, chlorhexidine, and diluted formocresol
solution).[3 ] Another study showed that diode laser irradiation is more effective than conventional
pulp capping technique.[4 ] Diode laser with wavelength ranging between 810 and 980 nm can be well absorbed
by hemoglobin and is suitable to decontaminate cavity and pulp coagulation in exposed
pulp.[5 ] Dentolaser 650 nm showed that irradiation of diode laser for 40 seconds in pulsed
mode increased fibroblast cell proliferation and odontoblast-like cells proliferation
in animal studies.[6 ]
[7 ]
Near-red laser spectrum with wavelength ranging approximately 630 to 675 nm or low-level
laser therapy (LLLT) is often used in medical fields because of its anti-inflammation
effect, analgesic, and biostimulation. LLLT is also capable to promote wound healing.[5 ]
[6 ]
[7 ] Several advantages of laser irradiation in vital pulp therapy compared with conventional
technique are decontamination effect, hemostatic effect, and biostimulation effect.[4 ] Previous studies concluded that red or near-red laser (600–1,200 nm) has biological
effect,[8 ] and biostimulator effect with energy density 0.05–10 J/cm2 can promote proliferation, and laser with energy density >10 J/cm2 can promote antiproliferation.[9 ]
Healing stages in reparative dentinogenesis consist of four steps which are moderate
inflammation, cell progenitor recruitment, cell progenitor proliferation, and final
differentiation.[10 ] Pulp is an important part in reparative dentinogenesis because dental pulp acts
as growth factor reservoirs. Vascular endothelial growth factor (VEGF-A), transforming
growth factor-1 (TGF-1), fibroblast growth factor-2 (FGF-2), bone morphogenic protein
(BMP), are the growth factors that play a significant role. These growth factors act
in regulation of progenitor cells' recruitment, cell proliferation, and dentine-secreting
cells differentiation.[1 ]
Growth factor and cytokine are key of molecule signaling which controlled and regulated
cellular involved in growth, homeostatic, and tissue recovery in dental pulp. Growth
factors are peptide molecules responsible for signaling some cellular process that
was happened after injury. Growth factors serve as signal transmitter of cell function,
as stimulator or inhibitor of growth and also as differentiation modulator. Growth
factor will regulate gene to control proliferation, cell differentiation, or cell
secretory product.[11 ] VEGF-A is growth factor for dental development, angiogenesis in dentin pulp complexes,
and proliferation in pulp recovery and dentin bridge formation.[6 ]
[12 ] TGF-β1 acts in homeostatic and tissue repair.[13 ]
[14 ]
Diode laser irradiation as alternative treatment option for vital pulp therapy has
no clear standardization about laser wavelength or exposure time. Based on that issue,
in vivo study has done to verify whether diode laser irradiation is effective for vital pulp
therapy with parameter of VEGF-A and TGF-β1 expression. Aim of this study is to prove
that the effect of diode laser 650-nm irradiation to the expression of VEGF-A and
TGF-β1 plays important roles in dental pulp-regulating cell proliferation, differentiation,
and revascularization.
Materials and Methods
Experimental Design and Ethics Approval
The experimental design in this study was post-test-only control group design. The
animal study (in vivo) was conducted in accordance with Government Regulation of The
Republic of Indonesia Number 95 of 2012 Concerning Veterinary Public Health and Animal
Welfare. Sample of the study was Rattus norvegicus male Wistar strain aged 8 to 12 weeks with an initial body weight of 200 to 250 g.
All samples were in healthy conditions with a total sample count of 48 for eight groups
(r = 6). All procedures performed in this study are ethical and have been approved by
the Ethics Commission of the Faculty of Dentistry, Airlangga University with approval
no. 244/HRECC. FODM/V/2020.
Laser Irradiate Preparation
The lasers used is a 650-nm diode lasers (Dentolaser 650, UNAIR, Indonesia), which
has been calibrated, with a standard irradiation distance of 1 cm with a power of
22 mW, considering the area of irradiation and controlling light at the time of caliber
([Fig. 1 ]). Laser is a device that irradiates light through a process of amplification which
is stimulated by photon emission. Diode laser works by releasing energy in the form
of photon as a result from combination of electron and electron hole in the device.
This photon will be absorbed by chromophore in the cell that causes changes in cellular
level such as cell proliferation.[4 ]
[15 ]
Fig. 1 (A–D ) Illustration of diode laser 650-nm irradiation with 1-cm distance for 40 seconds.
(adapted from Komabayashi et al).[10 ]
Pulp Perforation and Laser Irradiation
Experimental animals were anesthetized with Ketamine HCl (0.2-cc per kg body weight)
prior to preparation of a class-1 cavity on the occlusal surface of the maxillary
right first molar using a low-speed round diamond bur with a diameter of 1 mm to approach
the pulp chamber. Pulp roof perforation was performed using KFile no. 08 and was marked
by visually observed bleeding. The treatment group was divided into eight groups consisting
of a control group and a treatment group. that the control group perforated the pulp
roof and directly filled with GIC, and the treatment group irradiated with a laser
for 40 seconds and then filled with GIC after perforation of the pulp roof. Each group
was observed on different observation days, that is, days 7 and 14, and each was observed
to see the expressions of VEGF-A and TGF-β1.
Immunohistochemistry Examination of Vascular Endothelial Growth Factor-A and Transforming
Growth Factor-β1
Experimental animals from each treatment group to be treated by peritoneal injection
after 7 and 14 days from the treatment. After decapitation, the jawbone in the interdental
area of the maxillary right first molar was taken. Histological preparations were
made through the process of fixation, dehydration and infiltration, purification,
paraffin infiltration, embedding, sectioning, and sticking to the object glass. The
preparations are then checked to see if the tissue cuts made are right at the perforation
location. The next step was immunohistochemical examination using the monoclonal antibody
anti-VEGF-A antibody (11B5) ab38909 (Abcam) and anti-TGF-β1 antibody (TB21) ab27969
(Abcam). The slide was blocked with 3% H2 O2 in phosphate-buffered saline (PBS) incubation for 20 minutes and at room temperature.
Slides were washed with PBS pH of 7.4 and blocked with 1% bovine serum albumin (BSA)
in PBS for 60 minutes. Slide was labeled with a primary antibody anti-VEGF-A or TGF-β1
in 1% BSA overnight at 4°C. Slides were washed with PBS pH of 7.4 thrice for 5 minutes.
The slide labeled with secondary antibody goat antirat immunoglobulin (Ig)-G biotin
for 1 hour at room temperature. The washing was done thrice for 5 minutes with PBS
pH of 7.4. Slide incubation was performed with SA-HRP (streptavidin-horseradish peroxidase)
1:500 for 40 minutes at room temperature. Slides were washed with PBS pH of 7.4 thrice
for 5 minutes. The slide was dripped with substrate chromogen DAB (diamino benzidine
tetrahydrochloride 3.3) for 20 minutes. Slides were washed with PBS pH of 7.4 thrice
for 5 minutes and proceeded with dH2 O thrice each for 5 minutes. Counterstain was performed with methyl green 1% at room
temperature. The slide was soaked with tap water for 5 minutes and dried overnight
at room temperature. Mounting and cover with a cover glass, and then observed with
a light microscope at ×400 and ×1,000 magnification, counting 10 fields of view.
Statistical Analysis
Data were tabulated and analyzed using SPSS statistical software for Windows, Version
23.0 (IBM SPSS Statistics for Windows, Version 23.0. Armonk, New York, United States:
IBM). Differences in the mean value of VEGF-A and TGF-β1 were analyzed statistically
by one-way analysis of variance (ANOVA) at a 95% significance.
Results
Effect of Laser Irradiation to Vascular Endothelial Growth Factor-A Expression at
Days 7 and 14
Immunohistochemistry (IHC) examination is done by counting cells that expressed VEGF-A
from each group. Data examination of VEGF-A expression on the dental pulp healing
was done at days 7 and 14 with immunohistochemical staining showed brown cell color.
The number of brown cells was calculated and compared between the control group and
laser irradiation. The using of laser irradiation with Dentolaser 650 nm on dental
pulp for 40 seconds showed VEGF-A expression on day 14 higher compared with day 7,
and control group (days 7 and 14) by IHC examination ([Fig. 2 ]).
Fig. 2 Immunohistochemical staining of VEGF-A expression (×400 magnification, Nikon H600L
microscope, DS Fi2 camera 300 megapixels). (A ) Fibroblast cells at control group day 7. (B ) Fibroblast cells at control group day 14. (C ) Fibroblast cells at laser group day 7. (D ) Fibroblast cells at laser group day 14. VEGF, vascular endothelial growth factor.
There was elevation of VEGF-A expression from days 7 to 14 in laser and control groups
([Fig. 3 ]). [Table 1 ] showed that there was a significant difference in the VEGF-A expression within group
(p = 0.001), but there was no significant difference in VEGF-A expression between control
group and laser in the days 7 (p = 0.092) and 14 (p = 0.092).
Table 1
Mean and standard deviation (SD) of VEGF-A expression in IHC examination
Group
VEGF-A (X ± SD)[a ]
p -Value[b ]
Control H7
5.5 ± 1.643
0.001
Control H14
7 ± 1.095
Laser H7
8 ± 2.000
Laser H14
10.33 ± 1.966
Abbreviations: IHC, immunohistochemistry; SD, standard deviation; VEGF, vascular endothelial
growth factor.
a
b
p -Value: significance level of 0.05.
Fig. 3 Difference of VEGF-A expression at observation day 7 and 14 in IHC examination. *Significantly
different with α = 0.05. IHC, immunohistochemistry; n.s, not significant; VEGF, vascular endothelial
growth factor.
Effect of Laser Irradiation to Transforming Growth Factor-β1 Expression at Days 7
and 14
Data examination of TGF-β1 expression on the dental pulp healing was done at days
7 and 14 with immunohistochemical staining showed brown cell color. The number of
brown cells was calculated and compared between the control group and laser irradiation.
IHC staining examination on dental pulp after treated with Dentolaser 650 nm shows
increasing TGF-β1 expression at day 14 compared with day 7 and control group (days
7 and 14; [Fig. 4 ]). Expression of TGF-β1 in control group showed no significant increasing between
days 7 and 14 (p = 0.228), even though in the laser group (p = 0.79). There was a significant difference on TGF-β1 expression between control
and laser groups (p = 0.000; [Table 2 ]). TGF-β1 expression is significantly higher in laser group compared with control
on day 7 (p = 0.015). At day 14, significant difference also found in laser group compared with
control group (p = 0.02; [Fig. 5 ]).
Fig. 4 Immunohistochemical staining of TGF-β1 expression (×400 magnification, Nikon H600L
microscope, DS Fi2 camera 300 megapixels). (A ) Fibroblast cells at control group day 7. (B ) Fibroblast cells at control group day 14. (C ) Fibroblast cells at laser group day 7. (D ) Fibroblast cells at laser group day 7.TGF, transforming growth factor.
Fig. 5 TGF-β1 expression at day 7 and 14 in IHC examination on pulp fibroblast. *Significant
difference with α = 0.05. IHC, immunohistochemistry; n.s, not significant; TGF, transforming growth
factor.
Table 2
Mean and standard deviation (SD) of TGF-β1 expression in IHC examination
Kelompok
TGF-β1 (X ± SD)[a ]
p -Value[b ]
Control H7
4.5 ± 1.871
0.000
Control H14
5.83 ± 1.722
Laser H7
7.83 ± 2.041
Laser H14
10 ± 1.789
Abbreviations: IHC, immunohistochemistry; SD: standard deviation; TGF, transforming
growth factor.
a
b
p -Value: Significance level of 0.05.
Discussion
Present study is similar with a study conducted by Alghamdi et al that stated laser
irradiation with red or near-red wavelength (500–1,200 nm) has biostimulator effect.
Laser with energy density 0.05 to 10 J/cm2 can induce proliferation and laser with energy density >10 J/cm2 can induce antiproliferation effect. This study was conducted using laser with energy
density 2.2 J/cm2 , so it can induce proliferation.[9 ]
In this study, it was found that the expression of VEGF-A and TGF-β1 increased significantly
in the laser group, both on days 7 and 14. This significant difference means that
the use of a 650-nm diode laser increases the expression of VEGF-A and TGF-β1. This
is in accordance with the theory that LLLT can increase the modulation of tissue repair
processes by stimulating cellular reactions such as migration, proliferation, apoptosis,
and cell differentiation. These results are also similar with studies using a laser
with a wavelength of 635 nm, where it was concluded that the use of lasers can increase
cell proliferation seen from the expression of VEGF-A and TGF-β1.[16 ]
[17 ]
LLLT, including a 650-nm diode laser, acts on the mitochondria of cells. LLLT stimulates
photochemical reactions in cells, a process called biostimulation or photo-biomodulation.
When photon light is absorbed by the chromophores inside the cell, the electrons in
the chromophores are excited and jump from low-energy orbits to high-energy orbits.
This stored energy can be used by the system for several cellular tasks. Diode lasers
increase adenosine triphosphate (ATP) production, modulate reactive oxygen species
(ROS), and induce transcription factors. Some of transcription factors are regulated
in changes in cellular redox reactions. These will cause protein synthesis that ends
with cell proliferation and migration, modulation of cytokines, growth factors, inflammatory
mediators, and increased tissue oxygenation. Some of them associated with AP1, cFos
and cJun heterodimers, nuclear factor kappa B (NF-kB), p53, activating transcription
factors/cAMP response element binding protein (ATF/CREB), hypoxia inductible factor
(HIF)-1, and HIF-like factor.[6 ]
[18 ]
Increased ROS activates transcription factors that cause upregulation of genes that
play a role in cell proliferation and migration, cytokine production, and growth factor.[6 ] The NF-kB pathway is activated by cytokine receptors, such as TNF-α and interleukin
(IL). Cytokines secreted by T-lymphocyte cells, which play a role in the process of
forming dentin bridge, are TNF as a proinflammatory cytokine and TGF as an anti-inflammatory
cytokine. The balance between the two cytokines is what affects the thickness of the
αβ dentin bridge formed. TGF can induce proliferation and differentiation of β-stem
cell pulp into odontoblast-like cells.[19 ]
[20 ]
The TGF-β/suppressor of mothers against decapentaplegic (SMAD) pathway is one of the
pathways that influences the proliferation and differentiation of odontoblast-like
cells. SMAD is a protein that is expressed in odontoblast-like cells. SMADs-2, -3,
and -4 are activated after TGF-β1 signaling. TGF-β1 induces phosphorylation of SMAD2
and SMAD3. SMAD3 is required by TGF-β1 to induce FGF-2.[15 ]
[17 ]
Under hypoxia conditions, VEGF expression increases due to the bond between HIF-1
which activates VEGF mRNA transcription. VEGF can also be induced from the influence
of IL-6, IL-8, endothelins, calcium ions, nitric oxide, and TGF-β. VEGF-A has several
receptors, one of which is VEGFR2. VEGF plays a role in the process of angiogenesis,
leading to increase permeability of blood vessel membranes, proliferation, and migration
of endothelial cells.[6 ]
[18 ]
VEGF-A expression on control group in day 14 is higher than VEGF-A expression in day
7. In theory of wound healing process, VEGF starts showing at day 3 after injury and
is upregulated until day 7. VEGF expression will decrease in day 13 and after 3 weeks
is getting normal.[17 ]
[21 ] This is similar to the result of this study in VEGF-A group, in control and laser
groups, VEGF-A expression is increasing from days 7 to 14. In treated with laser group,
the escalation of VEGF-A expression in laser group is higher than the escalation in
control group. This showed that laser irradiation can increase VEGF-A expression in
reparative dentinogenesis process.
The increase of TGF-β1 expression in laser group is bigger than the escalation of
TGF-β1 expression in control group which means TGF-β1 expression is increasing physiologically
in repair process after injury happens, but laser irradiation can accelerate the repair
process. Results of this study is linear to other study that using propolis and calcium
hydroxide as pulp capping agent. Those studies stated that TGF-β1 expression is higher
on day 14 compared with TGF-β1 expression on day 7, both in control and treatment
groups.[22 ] TGF-β1 is a multifunction cytokine that plays important role in repair process after
injury.[1 ] TGF-β1 acts in inflammation process, recruitment of cell progenitor, proliferation,
and cell differentiation. This study is limited to 14 days of observation where TGF-β1
expression is increased, similar with graphic of wound healing process. After that,
in differentiation stage, antifibrinogenic factor such as stratifin will be released,
so that avoid over healing and keloid.[21 ]
Conclusion
Diode laser 650 nm with 40-second irradiation time shows increment from days 7 to
14 reflecting increase in pulp healing by modulating VEGF-A and TGF-β1 expression
since days 7 to 14.
