Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
1
Granulation tissue enriched by aspirin and omega-3 fatty acids in healing
experimental periodontal lesion
Filip Hromcika,b
, JanVokurkaa,b,†
, Eduard Gopfertc
, Martin Faldynac
, Marketa Hermanovab,d,#
, Michal Kyrb,e,#
,
MonikaVicenovac,#
, Lydie Izakovicova Hollaa,b
Aims. Granulation tissue (GT) and specialized pro‑resolving mediators such as lipoxins and resolvins are key elements
in the successful resolution of periodontitis. Aspirin‑triggered lipoxins and resolvins are even more powerful than
their natural analogues. Their biosynthesis can be accelerated by omega-3 fatty acids. The aim of this study was to
evaluate the use of GT enriched by aspirin and omega-3 fatty acids during the surgical treatment of periodontitis in
an experimental animal model (rabbit).
Methods. In each of 24 rabbits, two experimental periodontal defects were created. In total, 47 defects were treated
with open-flap debridement and one of three procedures: (1) GT extracted and soaked with aspirin and omega-3 fatty
acids (ASA+OMEGA3 group); (2) GT soaked with saline (PLACEBO group); or (3) GT left untreated (CONTROL group).
Then, the GT was replaced in situ. Primary evaluated criteria were the probing pocket depth (PPD) and the clinical at‑
tachment level (CAL). Necropsies were harvested 2, 6, and 12 weeks after surgery.The samples were used for histological
and molecular biological assessment.
Results. A trend of greater PPD and CAL in the ASA+OMEGA3 group was observed at 6 weeks. However, there was no
significant difference between them. During the observation period, tissue levels of FGF-7, IL-1β and TIMP-1 showed
a statistically significant decrease (P<0.05). For the other variables, the ASA+OMEGA3 group was comparable with the
PLACEBO and CONTROL groups.
Conclusion. This experiment did not demonstrate the superiority of the proposed approach. However, the enriched
granulation tissue did not impair healing outcomes.
Key words: periodontitis, granulation tissue, oral surgery, inflammation mediators, lipoxin
Received: November 3, 2019; Revised: December 23, 2019; Accepted: January 22, 2020; Available online: February 11, 2020
https://doi.org/10.5507/bp.2020.003
© 2019 The Authors. This is an open access article licensed under the Creative Commons Attribution License
(https://creativecommons.org/licenses/by/4.0/).
a
Clinic of Dentistry, St. Anne’s Faculty Hospital, Pekarska 53, 656 91, Brno, Czech Republic
b
Faculty of Medicine, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic
c
Veterinary Research Institute, Hudcova 70, 621 00, Brno, Czech Republic
d
First Department of Pathology, St. Anne’s Faculty Hospital, Pekarska 53, 656 91, Brno, Czech Republic
e
Department of Pediatric Oncology, University Hospital Brno, Cernopolni 9, 613 00, Brno, Czech Republic
#
These authors contributed equally to this work
Corresponding author: Lydie Izakovicova Holla, e-mail: holla@med.muni.cz
Introduction
Periodontitis is a multifactorial inflammatory disease
that leads to the destruction of periodontal tissues, with
potential tooth loss. Its development is associated with
bacteria residing in tooth biofilms close to or underneath
the gingival margin1
. Major signs of periodontal inflammation
are periodontal pocketing, clinical attachment
loss, alveolar bone resorption, and tooth mobility. The
treatment of periodontitis lies mainly in eliminating dental
biofilm along with calculus from the afflicted tooth
surface both above and under the gingival margin and
long-term maintenance of a biofilm-free environment2
.
Until recently, healing of an inflammation, not only
periodontitis, was believed to involve a passive process. It
has been revealed that in the late stage of inflammation,
so-called resolution (termination of inflammation) occurs.
The anti-inflammatory pathways are not sufficient
for resolution to take place and ruling out the proinflammatory
pathways (i.e. with NSAIDs) is not enough for
tissues to heal. Resolution has been reported to be an
active process leading to the regeneration of tissue and
re-establishment of homeostasis3
.
It has been suggested that periodontitis may be attributable
to a lack of adequate resolution of inflammation
and hyperactivity of pro‑inflammatory cytokines4,5
.
Resolution is driven by a family of lipid molecules known
as specialized pro-resolution mediators (SPMs), such as
lipoxins and resolvins6
. These are naturally synthetized
in the late phases of inflammation7,8
. Their synthesis can
be triggered by aspirin, which results in more powerful
and stable analogues9
. Both lipoxins and resolvins are derived
from poly‑unsaturated fatty acids (PUFAs), which
are cleaved from the cellular membrane and abundant in
fish oil10
. The acetylation of cyclooxygenase-2 (COX-2)
by aspirin not only blocks the production of prostaglan-
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
2
dins and leukotrienes but also switches the activity of
this enzyme to acting as a lipoxygenase and, thus, allows
the synthesis of lipoxins without cell‑to‑cell interaction10
.
Additionally, acetylated COX-2 carries out the synthesis
of aspirin‑triggered resolvins or protectin D1 (ref.11,12
).
Granulation tissue (GT) possesses great healing capacity
and offers a large number of new vessels, fibroblasts
and macrophages. Also, periodontal GT was proven
to contain a substantial number of mesenchymal stem
cells (MSCs), which correlates with its regenerative poten-
tial13,14
. It has been shown that GT promotes regeneration
and resolution of inflammation in periodontitis15
.
Inducing the synthesis of resolvins and lipoxins by the
addition of aspirin and PUFAs to GT during open-flap
periodontal procedures might help to resolve periodontitis
and, thus, enhance, accelerate and shorten periodontal
therapy. The aim of the present study was to evaluate the
effect of the addition of aspirin and PUFAs to granulation
tissue on the healing of experimental periodontitis.
Materials and Methods
Ethical approval
The experiment was performed in compliance with
Act No. 246/1992 Coll. of the Czech National Council
for the protection of animals against cruelty and with the
agreement of the Branch Commission for Animal Welfare
of the Ministry of Agriculture of the Czech Republic.
The study was performed according to the ARRIVE
guidelines for reporting in vivo animal research
(Supplementary S1, Checklist) (ref.16
).
Study design
The study was performed as a controlled blinded trial
in an animal model. In twenty‑four rabbits, experimental
periodontitis was induced via wire ligature and inoculation
by P. gingivalis. A total of 47 defects were created.
One defect had to be excluded because of a fracture in
the first premolar during the experiment. Six weeks after
ligature placement, the ligature was removed and the
rabbit was subjected to open-flap surgery to remove the
granulation tissue (GT) and replace it in its original interdental
space after one of the three following procedures:
ASA+omega3 group – GT was soaked with fish oil (n-3
PUFAs) and aspirin; PLACEBO group (PL) – GT was
soaked with saline; CONTROL group (CO) – GT was left
untreated. Necropsies were harvested 2 weeks (8 rabbits,
15 defects), 6 weeks (6 rabbits, 12 defects) or 12 weeks
(8 rabbits, 16 defects) after surgery, and the samples were
preserved for histological and molecular biological assess-
ments.
Animals
In this experimental model, twenty-four specificpathogen
free 3-month old White New Zealand rabbits
(Oryctolagus cuniculus f. domesticus) (supplied by a local
production company approved by the Ministry of
Agriculture of the Czech Republic and registered as CZ
62760247) were enrolled, including both males and females.
They were kept at the Veterinary Research Institute
(Brno, Czech Republic) in experimental cages certified
by the Ministry of Agriculture of the Czech Republic (CZ
62760124) according to standard protocols corresponding
to Czech regulations (The Animal Protection and
Welfare Act No. 246/1992 Coll. of the Government of the
Czech Republic). The experiments were approved by the
Branch Commission for Animal Welfare of the Ministry
of Agriculture of the Czech Republic (permission number
22373/2016-MZE-17214).
Ligature-induced periodontitis
The ligature-induced periodontitis model described in
many studies17,18
was used to develop periodontal defects.
Fig. 1. A ligature was tied around the first mandibular premolar to promote gingival irritation and
induce experimental periodontitis.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
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The suspension of Porphyromonas gingivalis used for induction
was cultivated from a stock sample (CCM 3985,
isolated from a subcutaneous abscess) and adjusted to the
desired optical density (corresponding to a concentration
of 4.5‑6.5 x108
 CFU/mL).
Following anaesthesia by intraperitoneal injection of
ketamine and xylazine, an orthodontic steel wire of 0.012
inches in diameter was tied around the first mandibular
premolars on both the left and right sides as apically as
possible (Fig 1). A notch was created in the mesial cervical
area of the tooth with a small steel bur to improve retention
of the ligature. Moreover, the ligature was secured
by applying a small amount of light-cured flow composite
after etching the enamel with 37% phosphoric acid for
30 s. The epithelial junction around the first premolar
was disrupted with an elevator. When the ligatures were
fastened, two millilitres of P. gingivalis suspension was applied
to the gingival areas of both lower first premolars.
Such inoculation was repeated every day for 6 weeks to
establish experimental periodontitis. The ligatures were
removed 6 weeks after their placement, and experimental
treatment was performed.
Surgery, experimental treatment
Six weeks after ligature placement, experimental periodontitis
had developed, and a second intervention was
performed under general anaesthesia, the ligature was
removed, and open-flap surgery was performed on the
interdental space between the first and second mandibular
premolars. The interdental papilla on both buccal and
lingual aspects was elevated, and GT was extracted. At
this time, a treatment option was randomly chosen for
the actual defect. In the ASA+omega3 group, GT was
soaked for four minutes with acetylsalicylic acid at a
concentration of 81 mg/mL (Kardegic, Sanofi Winthrop
Industrie, Quétigny, France) and subsequently for another
four minutes with an omega-3 PUFA solution (Omega3
Forte, Walmark, Třinec, Czech Republic) containing 18%
eicosapentaenoic acid (EPA), 12% docosahexaenoic acid
(DHA) and vitamin E. In the PLACEBO group, GT was
only soaked with saline. In the CONTROL group, GT was
not treated. In all groups, the GT was placed back into
its original interdental space, and the papilla was sutured
with a single non‑resorbable suture (Resolon, Resorba,
Nürnberg, Germany). A split-mouth design was followed,
so that in a single animal, two defects were treated independently
with randomly assigned treatments.
The animals were euthanised 2 weeks (8 rabbits, 15
defects), 6 weeks (6 rabbits, 12 defects) or 12 weeks (8
rabbits, 16 defects) after the surgery (randomly assigned)
with a lethal dose of T61 (MSD Animal Health, Boxmeer,
Netherlands) applied intracardially under general anaesthesia.
Necropsies were then harvested.
Clinical evaluation
Clinically, the probing pocket depth (PPD) and clinical
attachment level (CAL) were measured with UNC
periodontal probe (Supplement S10) before surgery and
after necropsy at six locations around the second mandibular
premolar (Fig 2) to assess two adjacent interdental
spaces – P1-P2 and P2‑M1. Clinically, PPD was measured
as a distance from the gingival margin to the bottom of
the defect or gingival sulcus, respectively. For CAL measurement,
the most coronal point of the tooth enamel was
used as a reference because no cemento-enamel junction
is identifiable in rabbit teeth.
Histological analysis
Resected specimens of rabbit mandible were processed
and histopathologically examined at the 1st
Department of
Pathology, St. Anne’s University Hospital Brno. Resected
tissues were initially fixed in 10% neutral buffered formalin
for 48 h and were subsequently subjected to decalcification
in Decalcifier DC1 (LABONORD, Templemars,
France) solution containing formic acid and formalin at
room temperature. The decalcifying fluid was replaced
daily until the completion of decalcification. Tissue slices
including the first and second premolar, where the ligature
was placed, were used. These specimens were then
dehydrated, embedded in paraffin and sectioned along the
premolars in a mesiodistal plane. Four-micron-thick tissue
sections were stained with haematoxylin-eosin. Mounted
slides were histopathologically examined using a uniform
microscope and camera settings (Olympus BX43 and
PROMICAM 3-12C). The areas between the first and second
premolars were analysed under light microscopy. The
level of epithelialization of the defects, quality of connective
tissue, and presence of inflammatory infiltrates and
reactive osteoplasia were evaluated and scored (Table 1).
The size of the intrabony defect (distance of the alveolar
bone from the top of the defect) was measured using a
100 µm scale.
Molecular biological assessment
RNA isolation and reverse transcription
For RNA stabilization, thirty samples (< 3 mm) were
excised from the buccal portion of the interdental papilla
P1-P2 during necropsy. The excised tissue was directly imFig.
2. Measurement of primary observed parameters, probing
pocket depth (PPD) and clinical attachment level (CAL).
PPD and CAL were measured at six locations around the second
mandibular premolar before treatment (day 0) and on the
day of necropsy (2 weeks, 6 weeks or 12 weeks later).
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
4
mersed in RNAlater (Qiagen, Germany), then kept at 4 °C
overnight and finally stored at -70 °C until RNA isolation.
Prior to RNA isolation, the tissues were homogenized in
TRI Reagent RT (Molecular Research Center, Inc., USA)
with zirconia/silica beads using a MagNALyser instrument
(Roche, Switzerland). RNA was obtained by phase
separation using 4‑bromoanisole with15 min of centrifugation
at 4 °C and was further purified using an RNeasy
Kit (Qiagen, Germany) according to the manufacturers’
instructions for an animal tissue protocol. A sample of
the obtained total RNA in 18 µL of Qiagen RNase‑free
water was spectrophotometrically measured to determine
the concentration and purity in a NanoDrop 2000 UV-Vis
spectrophotometer (Thermo Fisher Scientific, Waltham,
USA). The same amount of RNA from every sample
(2.4 µg) was directly reverse transcribed using M‑MLV
reverse transcriptase (200 U, Invitrogen) and an oligo(dT)
RT primer at 37 °C for 1.5 h. Then, the cDNA was stored
at -20 °C until use. RNA integrity was checked by means
of electrophoresis.
qPCR
For qPCR purposes, the cDNA of each sample was
prepared in triplicate reactions in a 384‑well plate with the
assistance of a Nanodrop II liquid dispenser (Innovadyne
Technologies, Rohnert Park, CA). Each reaction mixture
with a total volume of 3 µL consisted of 0.5 µL of
5-fold diluted cDNA, 10 pmol of a gene-specific primer
pair (Generi Biotech, Hradec Kralove, Czech Republic,
Supplement S2) and 1.5 µL of master mix (QuantiTect
SYBR Green PCR Kit, Qiagen, Hilden, Germany) following
the manufacturer's recommendations. qPCR was
performed in a LightCycler 480 (Roche Applied Science,
https://www.roche.com/) under the following conditions:
denaturation (95 °C for 15 min) followed by 45 amplification
cycles (95 °C for 15 s, 58 °C for 30 s and 72 °C
for 30 s), and the quantification cycle (Cq) value was
obtained for each gene in each reaction. The gene expression
of 9 target genes and 3 housekeeping genes (HPRT‑1,
GAPDH, HMBS), as potential reference genes for the
normalization of data (Supplement S9), was measured.
Using the Excel add-in software NormFinder, HPRT‑1
was evaluated as the reference gene showing minimal gene
expression among all samples tested. HPRT-1 also served
as a positive internal control for qPCR. Specific primer
pairs were designed using the Primer-BLAST NCBI web
page. To prevent the detection of genomic DNA, residual
primers were designed to ensure that at least one primer
of a given primer pair spanned an mRNA exon junction.
Gene expression was calculated using the formula
[1/(2 Cqoftarget gene
)]/[1/(2 Cqofreferencegene
)] (ref.19
), including
adjustment to reference gene expression. Relative gene
expression was characterized as the ratio of the gene
expression of the sample under the tested experimental
treatment to that of the control sample without experimental
treatment. Melting curve analysis of the amplified
products was performed to test product specificity
using LightCycler 480 Software 1.5.0.39 (Roche Applied
Science; https://www.roche.com), and negative controls
were included during the entire process to monitor potential
nucleic acid contamination.
Statistical analyses
Because we had no prior information of possible
measurement results, we used the resource equation20
to estimate the number of animals. The optimal number
of investigational units for a simple analysis of variance
(ANOVA) design assumes the error degrees of freedom
(Error df = total df – treatment df) to be 10 to 20. In our
experiment, there were three treatments investigated, and
the animals were sacrificed at three time points. Each
animal underwent two investigational procedures. Thus, a
total 24 animals yield the Error df of 14 (24 animals yield
48 investigational units, 16 for each time point resulting
in 14 EDF given three treatments).
PPD, CAL and bone loss were evaluated using oneway
ANOVA. Differences between the initial value
(Day 0) and the second measurement (2 weeks, 6 weeks,
or 12 weeks) were tested separately for each defect, and
summaries of the results are displayed as error bar plots
(Fig. 3 and 4). Histological variables were analysed on a
semi-quantitative ordinal scale, and the results are shown
in bar plots. Differences between the analysed groups
were tested using the chi‑square test (Supplements S2S5).
Molecular data were logarithmically transformed
to achieve an approximately normal distribution.
Transformed molecular data were analysed using ANOVA
and displayed in error bar plots. Analyses were performed
using R 3.4.3 software21
. The level of significance was
considered alpha=0.05.
Table 1. Semi-quantitative classification of histologic parameters used for evaluation of the harvested samples.
Level of epithelialization Connective tissue Inflammatory infiltrate Reactive osteoplasia
1 Persistent defect 1 Immature, cellular,
granulation tissue
1 Intense, mixed,
intra- and subepithelial
1 Missing
2 Partially persistent
defect
2 Mature fibrous tissue 2 Moderate, mixed,
intra- and subepithelial
2 Present
3 Complete
epithelialization
3 Minimal, sparse
Scheme used for semi-quantitative evaluation of the level of epithelialization, quality of connective tissue, and presence of inflammatory infiltrates
and reactive osteoplasia.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
5
Fig. 3. Probing pocket depth.
Difference between baseline measurements (Day 0) of PPD at
locations 1 and 6 and PPD measured at 2 weeks, 6 weeks, or
12 weeks.
Fig. 4. Clinical attachment level.
Difference between baseline measurements (Day 0) of CAL at
locations 1 and 6 and PPD measured at 2 weeks, 6 weeks, or
12 weeks.
Results
No significant differences were found between groups
at the measured time points. The changes in variables are
described in detail below.
At two weeks after the surgery, in all groups, PPD and
CAL showed insignificant decline compared to the initial
value (Day 0) (Fig 3 and 4). A difference in CAL change
between the groups can only be seen for locations 2 and
5 (P=0.041). No other differences were observed between
the groups regarding PPD and CAL (Fig 3 and 4). No
significant differences were found for the molecular indicators
or histopathological criteria (Supplements S2-S6).
At six weeks, according to a still-declining PPD, the defects
tended to heal, or post-operative swelling per­sisted.
At the same time point, however, CAL increased in the
ASA+omega3 and PLACEBO groups, in contrast to the
decrease in CAL in the CONTROL group. Nevertheless,
no significant differences in the changes in PPD and CAL
were observed between the groups (Fig 3 and 4). However,
there was a significant difference in IL‑17 levels (P=0.05)
in the examined tissues, with the highest values being observed
in the PLACEBO group (Supplement S7i). For the
other molecular and histopathological parameters, no differences
could be confirmed at this time point.
At twelve weeks, both PPD and CAL returned to levels
close to baseline in all study groups. Indeed, PPD and
CAL almost reached baseline values (Day 0) at closer
measurement locations (1 and 6), nut there was no significant
difference between the groups (Fig 3 and 4). The
examined molecular markers did not show any difference
between groups at this time point (Supplement S7).
However, the difference in histologically evaluated bone
loss was found to be nearly significant (P=0.057), and the
observed bone loss was greater in the PLACEBO group
compared to the other study groups (Supplement S6).
In all the groups, bone loss was greater than at baseline.
During the observation period of 12 weeks, tissue
­levels of FGF-7, IL-1β and TIMP-1 showed a statistically
significant decrease over time (Supplement S8). This was
not the case for IL‑10, IL-17, MMP-9, TGFβ-1 and VEGF,
whose levels tended to remain constant, or for TNF-α,
which increased insignificantly.
Of the histopathological parameters, changes could
be observed in terms of an increasing grade of epithelialization,
maturation of connective tissue, and decline of
inflammatory infiltration (Supplements S2-S5). However,
the results did not prove to be significant.
The defects at different healing stages with their histological
characteristics are shown in Fig 5.
Discussion
In this study, we analysed the clinical, histopathological
and molecular biological parameters of the healing of
periodontal defects after treatment using three different
approaches. We used the ligature-induced periodontitis
model in rabbit.
The primary observed parameters, PPD and CAL,
showed rather contradictory results and rarely reached
the level of statistical significance. The measured PPD
values at 2 weeks showed a decline, meaning that the
defect was smaller than at Day 0. This difference might
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
6
Fig. 5. Defect healing stages and histological characteristics.
At 2 weeks (A,B), 6 weeks (C,D), and 12 weeks (E,F) after surgery (H&E, original magnification
x20 (A,C,E), and x100 (B,D,F). (A,B) Two weeks after surgery, the defect persists, with epithelialization
only observed at its periphery, and dense mixed inflammatory infiltrate is present at the
base of the wound and within the highly cellular granulation tissue. (C,D) Six weeks after surgery,
the defect is epithelialized, with moderate to intense mixed inflammatory infiltrates within the
epithelium and the underlying highly cellular immature connective tissue, and proliferation of
fibroblasts and capillaries is observed. (E,F) Twelve weeks after surgery, the defect is completely
epithelialized, with minimal subepithelial inflammatory infiltrate, and mature fibrous tissue is
present in the interdental area. E: epithelium; D: defect; GT: granulation tissue; ICT: immature
connective tissue; CT: connective tissue; BL: bone level.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019; 163:XX.
7
have been caused by oedema rather than healing of the
site. At 6 weeks, a negative difference was even more
noticeable, meaning that healed or still swollen tissues
impeded the access of the probe to greater depth. For
the ASA+omega3 group, this deviation was less obvious,
probably due to less post-operative oedema in this group.
However, at 12 weeks, PPD increased again as oedema
subsided and immature attachment allowed the probe to
penetrate deeper. At this point, PPD almost reached the
initial values measured at day 0.
On the other hand, CAL at 2 weeks corresponded
to the initial values in the ASA+omega3 and PLACEBO
groups, but not in the CONTROL group, where CAL increased.
At 6 weeks, the opposite situation was observed,
when the values increased above the initial levels in the
ASA+omega3 and PLACEBO groups, but a decline in
CAL was observed in the CONTROL group. We can attribute
this finding to the post-operative oedema, which
was similarly noticeable in the PPD values. Finally, at
12 weeks, the CAL values for all the groups returned to
near-baseline levels. Thus, the levels of CAL at 12 weeks
did not differ from the initial measurements, showing no
significant change in attachment position after the observation
period.
It is important to note when considering our results
that the artificial defects were created with an average
PPD of 2.6 mm and CAL of 5.6 mm (locations 1 and
6), and histological bone loss could not be examined
on day 0. The PPD and CAL varied by approximately
1 mm. The standard deviations of the PPD and CAL
changes were 1.1 mm and 1.5 mm, respectively. Using a
periodontal probe, the discriminating power is 1 mm, so
an experienced observer can measure differences with a
0.5 mm accuracy at best. All the above-mentioned facts
imply that we were dealing with relatively small changes
in the evaluated data compared to large data variability
and measurement uncertainty, which could be responsible
for the non-significant results. Other similar studies
performed in a ligature-induced periodontitis model have
never used PPD and CAL as primary outcomes. Instead,
bone loss17,22-25
or root exposure24
have predominantly
been measured clinically with a 0.5 mm scaled probe after
defleshing of necropsies or have been histologically
measured in stained samples. These studies use rats or
rabbits for their experiments. Or, if PPD was selected as
a primary criterion, a larger animal such as monkey was
employed26,27
. However, for our experiment, clinical parameters
PPD and CAL were of major interest.
Gradual decrease in the tissue levels of FGF-7 (also
known as keratinocyte growth factor), IL‑1β (pro-inflammatory
mediator) and TIMP-1 (metalloproteinase inhibitor)
corresponded to the healing of defects. It may be of
interest that MMP9 was maintained at a significant level
even 12 weeks after ligature removal, when tissue remodelling
is assumed to be complete.
Our approach includes the induction of pro‑resolution
pathways by aspirin and omega-3 PUFAs. Such interventions
in GT are assumed to oppose the pro‑inflammatory
phenotype of GT and result in better and faster healing
outcomes. Therefore, there might be important benefits
for healing in preserving and experimentally mani­pulating
GT in periodontal surgery. In the present study, we were
not able to prove such effect. Nevertheless, the enhanced
granulation tissue did not impair healing outcomes.
Conclusion
In this experimental rabbit model of ligature-induced
periodontitis, PPD and CAL cannot be used as primary
observed criteria. A trend to earlier healing was observed
when a periodontal defect was treated with GT soaked
with aspirin and omega-3 PUFAs. However, 12 weeks after
the intervention, the three groups did not exhibit any
significant differences. We can conclude that the enriched
GT may be returned into a periodontal defect with similar
treatment outcomes observed in the standard removal
of GT with periodontal surgery techniques. Our results
should be confirmed and further examined in a subsequent
clinical trial.
Acknowledgement: The study was supported by the
Ministry of Health of the Czech Republic (Project No.
NV16-28462A).
Author contributions: JV, LIH: conceptualization; FH,
MK: data curation; FH, MF, MV, MH: formal analysis;
JV, LIH: funding acquisition, project administration; FH,
JV, EG, MH: investigation; JV, FH: methodology; LIH: resources,
supervision; MK: software; FH, LIH: validation;
FH: visualization; FH, MV, MH, MK: manuscript writing;
LIH, EG, MF: writing - review and editing.
Conflict of interest statement: None declared.
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Supplemental Material: The online version of this article
(doi: 10.5507/bp.2020.003) offers supplemental material.