Lasers in Medical Science

https://doi.org/10.1007/s10103-019-02919-w

ORIGINAL ARTICLE

Evaluation of bradykinin, VEGF, and EGF biomarkers in gingival

crevicular fluid and comparison of PhotoBioModulation
with conventional techniques in periodontitis: a split-mouth
randomized clinical trial
Francesca Angiero 1 & Alessandro Ugolini 1 & Francesca Cattoni 2 & Francesco Bova 2 & Sergio Blasi 1 & Fabio Gallo 3 &
Gianguido Cossellu 4 & Enrico Gherlone 2

Received: 12 March 2019 / Accepted: 12 November 2019

# Springer-Verlag London Ltd., part of Springer Nature 2019

Abstract
Periodontal disease is a chronic progressive inflammatory process leading to damage of tooth-supporting tissues. This compar-
ative study assessed the effect of PhotoBioModulation (PBM) versus conventional therapy, and investigated biomarkers involved
in the healing process. The test group comprised twenty systemically-healthy non-smoking subjects with chronic periodontitis
with the presence of two matched contro-lateral premolar sites (probing depth > 5 mm); twenty subjects without chronic
periodontitis (CP) served as control group. Patients were treated at baseline, either with scaling and root planing (SRP group)
or with a procedure entailing SRP supported by PBM (PBM group). The laser used was a diode laser operating at 645 nm
wavelength, 10 J/cm2, and 0.5 W/cm2 with a 600 μm fiber optic. Crevicular fluid levels of bradykinin (BK), vascular endothelial
growth factor (VEGF), and epidermal growth factor z (EGF) were determined at both sites. Crevicular fluid specimens from both
groups were analyzed with the ELISA TEST. Clinical differences in analyzed outcomes were observed in favor of PBM
treatment. Taking average values as 100%, the reduction in BK concentration was 47.68% with SRP and 68.43% with PBM
on day 3; the VEGF concentration decreased by 35.73% with SRP and 48.59% with PBM on day 7; the EGF concentration
increased by 55.58% with SRP and by 58.11% with PBM on day 21.
Clinical parameters improved significantly in both groups (pooled mean values of probing depth decreased from 5.6 to 4.5
mm; gingival index from 1.92 to 1.1; and bleeding on probing from 49.67 to 23.23) but did not vary significantly between the
PBM and the SRP group. The results confirmed that PBM have beneficial effects in the early phases of the healing process
playing a role in modulation of BK, EGF, and VEGF in gingival crevicular fluid levels; both groups had significant clinical
improvement over control but there was no significant difference between them, only a trend for PBM group. The overall results
of the study suggest a potential benefit of PBM in conjunction with SRP in treating chronic periodontitis.

* Alessandro Ugolini Gianguido Cossellu

Alessandro.ugolini@unige.it gianguido.cossellu@unimi.it

Francesca Angiero Enrico Gherlone

francesca.angiero@unige.it gherlone.enrico@hsr.it

Francesca Cattoni 1
Department of Medical Sciences and Diagnostic Integrated,
cattoni.francesca@hsr.it University of Genoa, Genova, Italy
Francesco Bova 2
Dental School, Vita-Salute San Raffaele University, Milan, Italy
bova.francesco@hsr.it 3
Section of Biostatistics, Department of Health Sciences (DISSAL),
Sergio Blasi University of Genoa, Genoa, Italy
sergioblasi8@gmail.com 4
Department of Biomedical, Surgical and Dental Sciences, University
Fabio Gallo of Milan, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore
fabio9980@gmail.com Policlinico, Milan, Italy
 Lasers Med Sci

Keywords Periodontology . Low-level laser therapy . Wound healing . Dentistry . Effect of Lasers in Tissue . Lasers in Dentistry .
Lasers . Diode . PhotoBioModulation

Introduction after PBM treatment compared with periodontal sites treated

with SRP alone. The majority of clinical trials found no dif-
Periodontitis is a chronic inflammatory disease with multi- ference for other biomarkers, or report conflicting results, for
factorial etiology that affects the supporting structures of teeth, levels of TGF-β1, MMP-1, FGF [18], IL-1β [19], ILs, FGF,
causing loss of clinical attachment, alveolar bone resorption, PDGF, TNF-α, VEGF [20], tissue plasminogen activator,
periodontal pocketing, gingival inflammation, and tooth loss. and plasminogen activator inhibitor 1 [21].
Conventional periodontal therapy includes both surgical and Thus, the benefits of the PBM approach compared with
non-surgical approaches. Non-surgical therapy, which involves conventional treatment alone have yet to be clearly
instrumentation of the inflamed tissues to eliminate infection, established. The present study aimed to evaluate the effect of
arrest disease progression, and regenerate the compromised peri- PBM used as an adjunct to SRP on clinical parameters and on
odontium, is the primary recommended approach to control peri- bradykinin, vascular endothelial growth factor (VEGF), and
odontal infection. Scaling plus root planing (SRP) is the main epidermal growth factor (EGF) levels in the gingival crevicu-
technique used to debride periodontal pockets [1–5]. lar fluid (GCF) of subjects with chronic periodontitis.
Because conventional therapies entail wounding the al-
ready inflamed periodontal tissues, the consequence of these
procedures depends to a great extent on the cellular and mo-
Materials and methods
lecular events associated with wound healing.
Short-term management of the healing process is one of the key
The study was designed as a split-mouth randomized controlled
points in clinical practice. Research is thus ongoing to improve the
clinical trial. The study protocol was approved by the Local
effectiveness of SRP, reduce patient discomfort, and optimize treat-
Ethics Committee, Milan, no. PR246 and written informed con-
ment results, for example by means of the use of lasers [6].
sent was obtained from all study participants in accordance with
PhotoBioModulation (PBM) is known and recommended for
the Helsinki Declaration (1975; revised, 2002).
its pain-reducing, anti-inflammatory, and wound-healing effects.
Participants were recruited from among individuals apply-
The action of PBM is based on photo-biological processes, rather
ing consecutively to the Department of Dentistry San Raffaele
than cutting or ablating the tissues [7, 8]. Known also as soft laser
Vita-Salute, Milan. Inclusion criteria were: > 18 years old,
therapy, biostimulation, or photo-biomodulation, PBM has been
presence of ≥ 20 teeth, presence of generalized chronic peri-
shown to possess biostimulatory action on various cell types and
odontitis (CP), and presence of at least two matched contro-
tissues, through its ability to affect the mitochondrial respiratory
lateral premolars with 5–7 mm periodontal pocket depth (no
chain, thereby increasing adenosine triphosphate production,
systemic diseases and no use of antimicrobial medications
which in turn facilitates fibroblast proliferation, angiogenesis,
within the past 6 months prior to recruitment.
growth factor release, and collagen synthesis [9–12].
Exclusion criteria were subjects with systemic disease or who
Unlike the powerful surgical lasers that require > 1 W, the
were under medication known to affect inflammation and the
power of these lasers can be modulated to obtain parameters
wound healing process; subjects who had undergone periodontal
(fluence, power density) suitable for PBM, with wavelengths
treatment within the past 6 months; smokers; pregnant; or lactating.
usually in the red and near-infrared spectrum.
Periodontal status was assessed by clinical examination
Alternatively, a bank of light-emitting diodes (LEDs) can
following the classification proposed by the American
be applied directly on diseased tissues and used to change
Academy of Periodontology (AAP) 1999 [22].
intra-cellular photoreceptors [13]. Light absorption by the tis-
Twenty subjects (10 m, 10 f), mean age 38.25 ± 6.43, range
sues leads to a cascade of photo-biological events, which may
25–60 years, with CP, were included in the study; a further
have beneficial effects on periodontal healing [13, 14].
twenty healthy subjects (10 m, 10 f), mean age 35.46 ± 5.60,
Various cytokines play important roles in periodontal dis-
range 25–60 years, served as control group.
ease and wound healing (e.g., inflammation, proliferation,
and regeneration) that occur after periodontal treatment, but
relatively few clinical trials have investigated these parame-
ters, nor the benefits of PBM used as an adjunct to SRP. Few Treatment protocol
studies report significant differences in biochemical param-
eters in in vivo clinical trials, and only MMP-8 [15], TNF-α All patients received basic periodontal treatment, comprising
[16], IL-1β, and RANKL/OPG [17] appear to be reduced one-stage full-mouth scaling and root planing (SRP) with
Lasers Med Sci
ultrasonic scaler and Gracey Curettes, plus oral hygiene in-
structions, from a single clinician. After SRP, one of the two
premolars (left or right) was selected randomly (by coin toss)
to receive PBM (PBM group) or sham treatment (SRP group).
Sham treatment comprised simulated laser application, with-
out pushing the start button on the laser device. A dual wave-
length (980 nm–645 nm) diode laser device (Raffaello BIO
980 DMT S.r.l.–Italy: verified and calibrated by the manufac-
turer was used in all cases, operating at 645 nm wavelength.
The laser, working in continuous mode, was used in scanning
mode; irradiation was via a 600 μm fiber optic connected to a
probe with 0.5 cm2 output surface area, capable of emitting a
collimated gaussian beam with very low divergence. The
hand-piece was held perpendicular to the site under treatment,
at a distance of 1–2 mm, thus reducing any possible reflection;
the movement was “lawn-mowing” in the apico-coronal di-
rection. Irradiation was applied to each periodontal site at
baseline (the day of SRP), and after 1, 3, and 7 days.
Assuming the irradiated surface to be approx. 2 cm2, with
application time of 80 s for each site, and output power set at
250 mW, average fluence was 10 J/cm2 and average power
density (at the target) was 0.125 W/cm2 with a cumulative
dose of 20 J per site for each treatment (max n. of treatment
4×20J = 80 J).
Periodontal examination and GCF sampling
Clinical measurements and GCF sampling at baseline and
during follow-up appointments were performed by a single
clinician, blind to which side had received active laser treat-
ment. Baseline measurements of the probing pocket depth
(PPD) at six sites per tooth (mesio-buccal, mid-buccal, disto-
buccal, mesio-lingual, mid-lingual, and disto-lingual loca-
tions) using a UNC-15 probe, gingival index (GI), and bleed-
ing on probing (BOP) were recorded prior to SRP and 3
months after treatment.
The site for GCF sampling was first treated to remove
any supragingival plaque, isolated with cotton rolls, and
gently dried with an air syringe. GCF was sampled by
placing a prefabricated filter paper (Periopaper; ProFlow,
Inc., Amityville, NY, USA) in the crevice until mild re-
sistance was felt, and leaving it in position for 30 s. Any
samples contaminated with blood were discarded. GCF
samples were placed in sterile polypropylene tubes and
stored at − 80 °C until analysis; samples were taken be-
fore treatment and at different times according to each
variable laboratory protocol. Bradykinin (pg/ml) follow-
up was set 3 days after T0, VEGF (pg/ml) follow-up was
set 7 days after T0, and EGF (ng/ml) follow-up was set 21
days after T0.
GCF analysis
To analyze bradykinin, VEGF, and EGF, a commercial ELISA
(enzyme-linked immunosorbent assay) tests with rabbit anti-
IgG pre-varnished on 96 wells plate (12×8) was used
(abcam® and Sigma Aldrich) following the supplier’s instruc-
tions. Briefly, solutions (standard or conjugated to biotin) of
bradykinin, VEGF, and EGF were applied to the plate. The
plates were incubated at ambient temperature and for each test
procedure, polyclonal antibody of bradykinin, VEGF, and
EGF, respectively, was added to the plate. The plates were
again incubated at ambient temperature and then washed to
remove non-linked reagent, and streptavidin HRP(prepared
by adding 250 μl of deionized water to kit reagents) was
added. After again incubating at ambient temperature, surplus
reagents were removed by washing and the substrate TMB
(3,3′,5,5′-Tetramethylbenzidine) catalyzed by HRP was added
to generate a blue color. After incubation at ambient temper-
ature, a stop solution (HCl solution) was added to change the
color from blue to yellow. The intensity of the yellow colora-
tion is inversely proportional to the quantity of specific anti-
body captured on the plates.
Statistical analysis
The sample size was established considering BOP% as the
primary outcome variable. According to the results of the
power analysis, a sample size of 80 quadrants in 20 partici-
pants was identified for 80% statistical power, b = 0.20, and a
= 0.05 (to detect D = 10%).
Continuous variables are given as means with standard
deviations (SD) and categorical variables as number of
subjects and percentage values. Baseline clinical value
differences were evaluated using t test. In order to evalu-
ate the effect of used treatment (PBM or SRP), the linear
mixed model [23, 24] (LMM) was performed on log-
transformation of GI, probing pocket depth, BOP, brady-
kinin, VEGF, and EGF. The patient sampling units were
considered to be random factor in each LMMs and the
likelihood ratio test was then used to test the association
between the outcomes and the independent variables. The
differences, with an estimated p value lower than 0.05,
were selected as significant and data were acquired and
analyzed in R 3.5.1 software environment. Five subjects
were evaluated twice for the intraexaminer reproducibili-
ty; PPD values were recorded at the first visit and 3 days
before any treatment was performed. The interclass corre-
lation coefficient values for the overall intra-examiner
agreement were 0.965 (95% CI: 0.927–0.984; p <
0.001). The method error was considered negligible.
 Lasers Med Sci

Results Table 1 Longitudinal differences. The results of GI, probing pocket

depth, BOP, bradykinin, VEGF, and EGF are expressed as mean with
standard deviation. MR (95% CI): mean ratio with 95% confidence
All subjects completed the treatment and reported regularly interval; p value: likelihood ratio p value
for follow-up appointments; a total of 200 colony-stimulating
factor (CSF) samples from 40 subjects were collected, with no Descriptive statistics Mixed model
dropouts occurring during the study. GI MR (95% CI) p value
Baseline differences were observed for BOP clinical value (p 1.53 (0.4)
value < 0.0001). No significant clinical differences were ob- Time < 0.0001
0 1.92 (0.18) 1
served for BOP, probing pocket depth, and GI between SRP 1 1.17 (0.13) 0.61 (0.59:0.64)
and PBM groups. The means GI, probing depth, and BOP pa- Group 0.1242
rameters, reported in Table 1, were quite similar among groups. SRT 1.56 (0.41) 1
LLLT 1.51 (0.4) 0.97 (0.92:1.01)
Taking the parameters separately, it was found that:
Probing pocket depth (mm) MR (95% CI) p value
5.1 (0.7)
& For bradykinin, there was a significant difference between Time < 0.0001
0 5.6 (0.4) 1
baseline SRP and baseline PBM (Wilcoxon Test-P <
1 4.5 (0.3) 0.81(0.78:0.83)
0.01); Group 0.3639
& For VEGF, there was a significant difference at baseline SRT 5.0 (0.6) 1
between SRP and PBM (Paired T Test P < 0.01); PBM 5.1 (0.7) 1.01(0.98:1.05)
BOP (%) MR (95% CI) p value
& For EGF, there was a significant difference between base- 36.47 (22.58)
line SRP and baseline PBM (Paired T Test P < 0.01) Time < 0.0001
0 49.67 (23.49) 1
1 23.26 (11.1) 0.48(0.37:0.64)
No significant GI, probing depth, and BOP differences be- Group 0.0877
tween groups were observed during the follow-up (Table 1: p- SRT 41.14 (25.48) 1
values: 0.1242, 0.3639, and 0.0877, respectively). Significant PBM 31.79 (18.41) 0.79(0.59:1.04)
Bradykinin (pg/ml) MR (95% CI) p value
reductions were observed between pre- and post-values of GI, 157.1 (123.43)
probing depth, and BOP (Table 1: p values < 0.0001). In partic- Time < 0.0001
ular, the GI, probing depth, and BOP had statistically significant 0 221.29 (131.51) 1
1 92.82 (71.4) 0.4(0.36:0.45)
increases of 39%, 19%, and 51% at the time 1, compared with Group < 0.0001
those measured at the time 0 (Table 1; MRs: 0.61, 0.81, and 0.79, SRT 168.54 (121.47) 1
respectively). The mean concentrations of bradykinin, VEGF, PBM 145.57 (125.83) 0.77(0.69:0.87)
VEGF (pg/ml) MR (95% CI) p value
and EGF in healthy subjects were 221.29 (SD = 133.23), 99.72
78.7 (22.56)
(SD = 6.45), and 836.85 (SD = 164.19), respectively. Time < 0.0001
Significant differences were found between the two treatment 0 99.72 (6.37) 1
groups (SRP vs. PBM) for bradykinin, VEGF, and EGF during 1 57.68 (9.18) 0.57(0.55:0.59)
Group < 0.0001
the follow-up (Table 1; p values < 0.05). In particular, bradykinin SRT 81.91 (19.57) 1
and VEGF were almost 23% and 10% lower in subjects with a LLLT 75.5 (25.04) 0.90(0.87:0.93)
PBM treatment than in subjects with SRT treatment (MR = 0.77 EGF (ng/ml) MR (95% CI) p value
1389 (603.36)
and MR = 0.90, respectively), while EGF concentration was Time < 0.0001
almost 3% higher in subjects with a PBM treatment than in 0 836.85 (162.07) 1
subjects with SRT treatment (MR =1.03). 1 1940.92 (293.32) 2.34(2.27:2.41)
Group 0.0255
Expressed in percentage terms, taking average pre-treatment SRT 1360.42 (574.55) 1
values as 100%, there was a decrease in bradykinin levels of LLLT 1417.35 (636.91) 1.03(1:1.06)
47.68% in the SRP group and of 68.43% in the PBM group; for
VEGF, there was a decrease of 35.73% in the SRP group com-
pared with 48.59% in the PBM group; for EGF, there was an
increase of 25.1% in the SRP group compared with 38.7% in the treatment of chronic periodontitis. The results confirmed
the PBM group. that the use of the laser promotes significant clinical improve-
ment (PPD, GI, and BOP). The use of PBM showed also
significant differences considering biochemical values (BK,
Discussion VEGF, and EGF) compared with the control group treated
with SRP alone.
In this split-mouth study, we compared the clinical and bio- During recent years, the use of lasers in the medical field is
chemical results obtained with PBM as an adjunct to SRP for becoming consolidated. Low-level lasers are currently in use
Lasers Med Sci
in general medicine to improve wound healing. CP treatment
primarily comprises non-surgical periodontal therapy, which
is a well-established procedure for treating the disease and
controlling its progression; PBM has recently been proposed
as an adjunctive treatment to optimize the outcome of CP
treatment.
The present split-mouth study aimed to evaluate the
effect of PBM as an adjunct to non-surgical treatment of
CP. To the best of our knowledge, this is the first study to
show that PBM in conjunction with scaling and root plan-
ing can significantly affect levels of bradykinin, VEGF,
and EGF in gingival tissues of patients with periodontal
disease in short-term follow-up. The importance of resolv-
ing the inflammatory phase has been widely reported,
suggesting that faster reduction of inflammatory media-
tors may be related to a better healing process.
Bradykinin derives from high molecular weight kininogen
due to the action of plasma kallikrein [25]. It is involved in
inflammatory processes and affects blood vessel dilation and
vascular permeability, as well as the initiation of pain; brady-
kinin levels have been reported to play an important role in
various inflammatory diseases, including allergic rhinitis,
asthma, and arthritis, and it is also thought to play an impor-
tant role in the pathogenesis of gingival inflammation and
bone resorption [25, 26].
VEGFs are a super-family of growth factors including
VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and pla-
cental growth factor. The VEGF-A isoform is the most repre-
sented in the healing process [27]. It is produced by endothe-
lial cells, keratinocytes, fibroblasts, smooth muscle cells, neu-
trophils, macrophages, and platelets. VEGF binds two recep-
tors of the tyrosine kinase family Flt-1 (VEGFr-1) and KDR
(VEGFr-2) expressing its function on proliferation, migration,
and differentiation of endothelial cells and chemotaxis [27,
28]. VEGF levels were evaluated in only one clinical trial,
comparing the effects of PBM as adjunct therapy to SRP, in
accordance with the present results, a significant reduction
after 7 days. In contrast with the present results, that study
found no difference between SRP and PBM groups [6, 20].
EGFs are a super-family of growth factors, including EGF,
HB-EGF, epiregulin, amphiregulin, neuregulin1-6, and
betacellulin. EGF binds tyrosine kinase receptor HER1,
HER2, HER3, and HER4, causing epithelialization, prolifer-
ation, and migration of keratinocytes. Epithelial wound
healing is in part mediated in an autocrine manner by EGF.
It induces a mitogenic response in human periodontal liga-
ment cells, which appears to be associated with the rapid
and selective activation of the Erk1/2 pathway [29].
Although levels of these markers were improved, they
failed to achieve levels comparable with those of the healthy
control group; this may be explained by considering that in
subjects with CP there was still the presence of a pathological
PPD (> 3 mm).
However, clinical parameters were positively improved in
both groups, and no relevant difference was revealed between
the two treatment groups. These results are in accordance with
a number of studies, which confirmed in long-term follow-up
that PBM has no adjunctive effect for periodontitis treatment
[17, 19, 21, 30–32]. However, they are in contrast with other
studies that have reported significantly better clinical out-
comes when PBM was used as an adjunct to SRP [15, 18,
20, 33–35]. The difference between the present study and
previous studies in terms of clinical outcomes might be due
to the use of different laser protocols and different clinical
baseline conditions.
Low-level lasers are thought to work by the interaction of
light with cells and tissue. This interaction might be affected
by some parameters, such as wavelength, power, energy den-
sity, treatment duration, treatment intervention time, method
of application, structure, and condition of tissue.
The dose applied during laser application is one of the
important treatment parameters determining benefit from
PBM. However, it is still not clear which are the optimal
parameters for PBM, due to the dearth of adequate studies; a
specific recommended dose has not yet been determined.
Clinically, evaluation of the number, type, depth, and devel-
opment of periodontal pockets is the basis for the diagnosis of
periodontitis. This research analyzed and compared the peri-
odontal index of both treatment groups (SRP group and PBM
group) and the control group. The results showed that, despite
the reduction of plaque index (PI) over the 3 months of the
study, there were no statistically significant differences.
Therefore, the two treatment groups showed a considerably
better improvement rate than the control group.
Conclusion
The overall results of the present study suggest that the appli-
cation of PBM as an adjunct to non-surgical periodontal treat-
ment may help to reduce inflammation, with significant im-
provement of bradykinin, VEGF, and EGF levels and of the
clinical parameters promoting the healing of periodontal tis-
sues in patients with CP.
However, further controlled clinical studies conducted on a
larger scale and with well-defined protocols are still need in
order to clarify the effects and establish an optimized protocol
for the use of PBM.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval The study was approved by the Local Ethics
Committee, Milan, no. PR246.

Informed consent Written informed consent was obtained from all
study participants in accordance with the Helsinki Declaration (1975;
revised, 2002).
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