UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3680
1
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
Eficácia da combinação de plasma rico em plaquetas liofilizado e ácido hialurônico após cicatrização cirúrgica periodontal:
um estudo experimental em modelo animal
Pitu WULANDARI
1
, Denny SATRIA
2
, Martina AMALIA
1
, BUDI
3
, Rio SIMANJUNTAK
3
1 - Universitas Sumatera Utara, Faculty of Dentistry, Department of Periodontics, Medan, Indonesia
2 - Universitas Sumatera Utara, Department of Pharmaceutical Biology, Medan, Indonesia
3 - Universitas Sumatera Utara, Faculty of Dentistry, Periodontics Residency Program, Medan, Indonesia
How to cite: Wulandari P, Satria D, Amalia M, Budi, Simanjuntak R. Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing: an experimental study on animal model. Braz Dent Sci. 2023;26(2): e3680.
https://doi.org/10.4322/bds.2023.e3680
ABSTRACT
Objective: the study aimed to investigate the effectiveness of the combined application freeze-dried platelet-rich
plasma (FD-PRP) and hyaluronic acid (HA) in healing after periodontal surgery. Material and Methods: This is an
experimental laboratory research with a design pre and post-test only control group design. Freeze-dried platelet-rich
plasma and HA were applied at periodontal surgical sites in periodontitis induced Wistar rats’ mandibulary incisors.
Bleeding on probing (BOP) was observed as a clinical aspect.
Porphyromonas gingivalis
was examined and counted.
Meanwhile, the healing process was seen by examining broblasts and Platelet-Derived Growth Factor-BB (PDGF-BB)
in histology and immunohistochemistry. In this study, BOP decreased faster after applying FD-PRP and HA. Data
were processed by ANOVA and Kruskal-Wallis test. Results: Clinical examination by measuring BOP level showed
better inammation resolution at 14
th
day (p=0.007).
Porphyromonas gingivalis (P. gingivalis)
count was also
reduced in the treatment group (p=0.001 at 3
rd
and 14
th
day). The histology aspect by measuring broblast count
(p=0.025 at 3
rd
day and p=0.001 at 14
th
day) and immunohistochemistry by measuring PDGF-BB expression
(p=0.014 at 3
rd
day and p=0.030 at 14
th
day) showed better tissue healing. Conclusion: combination of FD-PRP and HA
has accelerated the healing of periodontal surgical wounds.
KEYWORDS
Fibroblast; Hyaluronic acid; Periodontitis; Platelet-rich plasma;
Porphyromonas gingivalis
.
RESUMO
Objetivo: o estudo teve como objetivo investigar a ecácia da aplicação combinada de plasma rico
em plaquetas liolizado (FD-PRP) e ácido hialurônico (AH) na cicatrização após cirurgia periodontal.
Material e Métodos: Trata-se de uma pesquisa experimental com um delineamento pré e pós-teste apenas
de grupo controle. Plasma rico em plaquetas liolizado e AH foram aplicados em sítios cirúrgicos periodontais
induzidos por periodontite em incisivos mandibulares de ratos Wistar. Sangramento à sondagem (SS)
foi observado como aspecto clínico.
Porphyromonas gingivalis
foi examinado e contado. Enquanto isso,
o processo de cicatrização foi observado por exame de broblastos e fator de crescimento derivado de
plaquetas-BB (PDGF-BB) em histologia e imuno-histoquímica. Neste estudo, o SS diminuiu mais rapidamente
após a aplicação de FD-PRP e AH. Os dados foram processados por ANOVA e teste de Kruskal-Wallis.
Resultados: O exame clínico do nível de SS mostrou melhor resolução da inamação no 14º dia (p=0,007). A contagem
de
Porphyromonas gingivalis
(
P. gingivalis
) também foi reduzida no grupo de tratamento (p=0,001 no 3º e 14º dias).
2
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
O aspecto histológico pela contagem de broblastos (p=0,025 no 3º dia e p=0,001 no 14º dia) e imunohistoquímico
pela contagem da expressão de PDGF-BB (p=0,014 no 3º dia e p=0,030 no 14º dia) mostraram melhor cicatrização
tecidual. Conclusão: a combinação de FD-PRP e AH acelerou a cicatrização de feridas cirúrgicas periodontais.
PALAVRAS-CHAVE
Fibroblasto; Ácido hialurônico; Periodontite; Plasma rico em plaquetas;
Porphyromonas gingivalis
.
Streptococcus agalactiae
and
Streptococcus oralis
.
Importantly, PRP only exerts antibacterial properties
during activation and is effective against a wide range
of gram-positive and gram-negative bacteria [5,6].
Platelet rich plasma also contains leukocytes
which play a significant role as antibacterial.
The antibacterial effect of PRP is seen after
activation, however, PRP appears weak and
short-lived when used alone. Platelet rich plasma
acts synergistically with antibiotics and can be
considered as an adjunct treatment for pathogenic
infections especially in antibiotic-resistant cases [6].
The role of HA in periodontal disease is
through its anti-inammatory and anti-bacterial
effects. Tissue healing can be exploited using
collaboration in periodontitis therapy. Hyaluronic
acid is an extracellular part of connective tissue
that functions as a barrier from bacterial plaque
and fullls various extracellular functions that are
important in maintaining healthy gingival tissue.
It also has a multifunctional role in the wound
healing process and its mechanism is similar to
that of wound healing in periodontal tissues [7].
Hyaluronic acid is further involved in the activation
of inammatory cells such as polymorphonuclear
leukocytes (PMNs), macrophage functions,
including their migration to the wound site,
phagocytosis and killing invading pathogenic
microbes to prevent colonization and proliferation
anaerobic pathogens in the gingival crevice and
adjacent periodontal tissues [8]. Srisuwan et al. [9]
demonstrated that HA has potential in directed
tissue development, and this material is engineered
for various medical interests.
The study aimed to investigate the effectiveness
of the combined application freeze-dried platelet-rich
plasma (FD-PRP) and hyaluronic acid (HA) in
healing after periodontal surgery. A combination
of the use of PRP and HA in the biofunctional
scaffold can provide many advantages in wound
healing through the speed of healing compared to
advanced dressings, and the use of PRP alone as a
biostimulator or HA as a scaffold and this will also
reduce postoperative pain. Combination of the two
is expected to be faster in closing the wound with
the best esthetics [10].
INTRODUCTION
One of the critical aspects of the wound healing
response in the periodontium is the regeneration of
the cementum-connected collagen bers that cover
the tooth roots, gingival connective tissue and
alveolar bone. Immediately following injury to the
periodontal tissues, a vascular cascade is activated
to control local arteriolar and venous bleeding.
The platelet plug is formed by a mixture of brin
and bronectin which actively plays a mechanical
role in supporting and as a necessary framework
for tissue healing. Through the granules, locally
activated platelets will release growth factors,
cytokines and chemokines that will stimulate cell
proliferation, adhesion and migration. Platelet rich
plasma (PRP) is an autologous platelet containing
small volumes of plasma, which is enriched with
growth factors and proteins that stimulate cellular
processes such as chemotaxis, mitogenesis, cell
differentiation and angiogenesis [1,2].
Platelets rich plasma have shown benecial
effects by relieving postoperative discomfort
and preventing infection, as well as acting as
anti-inammatory and anti-bacterial. In recent
years, interest in the use of PRP in the treatment of
tissue infections has increased [3]. Although much
progress has been made in clinical practice, the
treatment of chronic wounds and bone infections
remains a challenge. Conventional treatments
for treating bone infections include debridement
of the infectious wound, use of antibiotics, and
restoration of the soft tissue covering the wound
infecting the bone. Often these treatments are
ineffective in producing a timely resolution of
infection, which can cause signicant morbidity
and an economic burden for the patient [4].
Bacterial attachment and growth is an
important initial step in colonization. Platelet
rich plasma can also inhibit the attachment and
growth of
P. gingivalis
and
Aggregatibacter
actinomycetemcomitans
which are periodontal
pathogens but platelet rich fibrin (PRF) cannot
kill these microorganisms. Platelet rich plasma
can inhibit the growth of microorganisms in vitro
such as
Enterococcus faecalis, Candida albicans,
3
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
MATERIAL AND METHODS
Experimental animals
This study tested the effectiveness of
combination FD-PRP and HA after periodontal
surgery healing in male Wistar rats, aged
8-12 weeks, weighing 200-250 grams and
having a healthy body condition, induced
by lipopolysaccharide bacteria
P. gingivalis
strain ATCC 33277. Calculation of the number
of samples using the Federer formula with a
total sample of 34 rats. This research has been
approved by the Research Ethics Commission
of the Faculty of Mathematics and Natural
Sciences in Universitas Sumatera Utara
(Animal Research Ethics Committees/AREC)
with letter No. 0350/KEPH-FMIPA/2021.
Experimental animals were adapted and kept in
cages placed in a room with sufcient airow and
light for 10 days. During the adaptation period,
the animals were monitored for food and drink
intake, urination, weight gain or loss and general
behavior of the animals to evaluate the stress
level of the animals. Animal cages are cleaned
every day of dirt and food residue. Animals were
fed food and water three times a day during the
trial period.
Periodontitis induction
Before doing periodontal treatment,
induction of periodontal pathogens was carried out
in experimental animals so that the experimental
animals suffer from periodontitis.The procedure
begins with administering anesthesia via
intramuscular injection with a solution of 10%
Ketamine and 2% Xylazine (2: 1), 0.12 ml/100 g
body weight. The ligature was inserted into the
gingival sulcus of the lower incisors in an “8”
shape using a sterile non-resorbable silk thread
combined with a single wire, and the animals
were given a high carbohydrate diet. One week
later, the induction of
P. gingivalis
bacteria
ATCC 33277 was carried out in the gingival sulcus
area.
Porphyromonas gingivalis
is a periodontal
pathogenic bacteria that can cause periodontal
destruction. After ten days, swabs were taken for
microbiological culture examination to ensure the
mice had been infected with
P. gingivalis
bacteria.
The high-carbohydrate diet was stopped after
the mice were conrmed to have been infected
with
P. gingivalis
bacteria, and the ligatures were
released and ready to be treated.
Periodontal treatment
After the experimental animal suffers from
periodontitis, it is followed by the surgical stage.
The samples were divided into two negative
control groups, two positive control groups, and
six treatment groups where all groups had been
induced with
P. gingivalis
. The negative control
was not given treatment; the positive control
was given flap surgery only using the modified
Widman ap method and the other 3 groups were
given ap surgery with single and combined drug
applications. This procedure was carried out at the
Animal House, Faculty of Mathematics and Natural
Sciences in Universitas Sumatera Utara. The internal
bevel scallop incision to the cristae alveolar; the
ap is elevated. The ap can be thinned to allow
adaptation to the gingival density around the teeth,
and a sulcular incision is made in the gingival sulcus
to release connective tissue from the root surface.
The gingival margin and granulation tissue were
removed with a curette; then, root planing was
carried out before the material was added.
Hyaluronic acid used is HA 0.2% obtained
from preparations with the gengigel brand, while
the FD-PRP used is a product of the Biotechnology
Laboratory, Faculty of Dentistry in Universitas
Gadjah Mada. The manufacture of a combination
of FD-PRP and HA was carried out at the Integrated
Laboratory, Faculty of Pharmacy in Universitas
Sumatera Utara using a chitosan-based gel. All tools
that will be used for the manufacture of chitosan
hydrogel based gels were sterilized in an oven at
170°C for 1 hour. Aseptic preparation of the gel was
carried out in a laminar air ow cabinet, 2 gram of
chitosan was put into a stamper then added with
a 1% lactic acid solution that had been diluted as
much as 50 ml and stirred slowly to form a 4%
chitosan hydrogel using a magnetic stirrer [11].
Clinical, microbiological, histological and
immunohistochemical examinations
The clinical parameters examined were
BOP on days 3 and 14 using a periodontal probe
inserted into the gingival sulcus of the lower
incisor. In addition, a swab was also performed
on the gingival sulcus to assess the levels of
P. gingivalis
by counting the number of bacterial
colonies on days three and 14. Then the rats were
euthanized and the periodontal tissues were taken
and immersed in 10% formalin buffer for 2-4 hours.
Furthermore, the specimen was processed at
the Histology Laboratory, Faculty of Medicine
4
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
in Universitas Sumatera Utara to obtain
preparations with hematoxylin-eosin staining.
After that, the expression of is also observed
PDGF-BB by immunohistochemical staining.
Immunohistochemical quantitative analysis was
performed under a binocular microscope.
Statistical analysis
The data obtained from the study are quantitative
data in the form of clinical, microbiological,
histological and immunohistochemical parameters
before and after treatment on days 3 and 14. The
data were processed by ANOVA and Kruskal-Wallis
test to see the difference between the groups on the
third and fourteenth day.
RESULTS
Based on the results of microbiological
examination, the average score of the lowest
number of bacteria was found in the positive
control group on days 3 and 14. Statistically
it was shown that the treatment significantly
reduced the number of bacteria scores in all
groups on days 3 and 14 (Table I).
Based on histological examination, the highest
mean broblast count score was found in the AH
and FD-PRP combination group on day 3 and the
lowest on day 14. This indicates that the treatment
signicantly increased the broblast count score on
day 3 and 14 statistically (Table II).
Based on immunohistochemical examination,
the highest average PDGF-BB expression score
was found in the FD-PRP group and the AH and
FD-PRP combination on day 3. Statistically, there
was a significant difference in mean PDGF-BB
expression in all groups. The treatment caused
PDGF-BB expression to increase signicantly on
the 3rd day. However, on day 14, the highest
PDGF-BB expression score was seen in the negative
control group. Statistically, the treatment also
increased PDGF-BB expression on days 3 and 14
(Table III, Figure 1 and 2).
Based on clinical examination, the highest
average BOP score was found in the positive
control group on days 3 and 14. The lowest BOP
score was found in the FD-PRP group on day 3
and in all treatment groups on day 14. Results
of statistical analysis on day 3 showed that the
treatment did not signicantly reduce the BOP
score, but this was different on day 14 which
showed that the treatment signicantly reduced
the BOP score (Table IV).
Table I - Differences of the number of bacterial colonies on days 3 and 14.
Group
Number of bacterial colonies
Day 3 Day 14
x ± SD p value x ± SD p value
Control (-) > 300.00 ± 0.000
0.001
a
*
> 300.00 ± 0.000
0.001
b
*
Control (+) 149.667 ± 25.697 59.000 ± 16.093
AH > 300.00 ± 0.000 246.333 ± 45.621
FD-PRP > 300.00 ± 0.000 269.667 ± 33.946
AH + FD-PRP > 300.00 ± 0.000 130.667 ± 14.571
*significance p <0.05.
a
ANOVA test on the 5 groups on day 3
rd
.
b
ANOVA test on the 5 groups on day 14
th
.
Table II - Differences of the number of fibroblast on days 3 and 14.
Group
Number of fibroblasts
Day 3 Day 14
x ± SD p value x ± SD p value
Control (-) 96.067 ± 6.379
0.025
a
*
119.867 ± 10,504
0.001
b
*
Control (+) 108.467 ± 4.051 86.200 ±7.597
AH 112.000 ± 2.029 53.067 ± 9.453
FD-PRP 89.800 ±33,950 55.200 ±6.349
AH + FD-PRP 180.267 ± 28.759 43.67 ±3.402
*significance p <0.05.
a
Kruskall Wallis test on the 5 groups on the 3
rd
day.
b
ANOVA test on the 5 groups on day 14
th
.
5
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
Table III - Differences of PDGF-BB expression on days 3 and 14.
Group
PDGF-BB Expression
Day 3 Day 14
x ± SD p value x ± SD p value
Control (-) 2,000 ± 0.000
0.014
a
*
3.330 ±0.577
0.030
b
*
Control (+) 3,000 ± 0.000 3.000 ± 0.000
AH 3.670 ±0.577 2.000 ± 0.000
FD-PRP 4.000 ± 0.000 3.000 ± 0.000
AH + FD-PRP 4.000 ± 0.000 2.330 ±0.577
*significance p <0.05.
a
Kruskall Wallis test on the 5 groups on the 3
rd
day.
b
Kruskall Wallis test on the 5 groups on the 14
th
day.
Figure 1 - Immunohistochemical features of PDGF-BB day 3. A. Negative control group, B. Positive control group, C. AH group, D. FD-PRP group,
E. AH and FD-PRP combination group (magnification 400x).
Figure 2 - Immunohistochemical features of PDGF-BB day 14. A. Negative control group, B. Positive control group, C. AH group, D. FD-PRP group,
E. AH and FD-PRP combination group (magnification 400x).
6
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
DISCUSSION
This research is an experimental study with
a pre and post-test only group design. This design
was chosen to sensitize research subjects who might
give a different response compared to without the
pre-test [12]. Wistar rats were selected as research
samples because they are an ideal standard for
experimental animal studies that are representative
of mammals [13]. Murdiastuti et al. [14]
stated that the content of growth factors in the
collagen-activated FD-PRP had higher PDGF levels
than conventional platelet rich plasma.
Researchers in this study proved the
effectiveness of FD-PRP in combination with HA
in the healing of periodontal surgical wounds in
this article. Researchers found a decrease in the
level of
P. gingivalis
on day three and day 14 in
all treatment groups (Table I). This proves that
FD-PRP and HA have antimicrobial properties
against
P. gingivalis
. This is in accordance with
the research of Chen et al. [15] who proved that
hyaluronic acid has an antimicrobial effect against
P. gingivalis
. In another study, Pham et al. [16] also
stated that platelet rich plasma has antimicrobial
properties against
P. gingivalis
. Researchers
found FD-PRP in combination with HA to have
antimicrobial properties against
P. gingivalis
compared to FD-PRP or HA alone.
Based on histological examination in this
study, it was found that on the third day there
was an increase in the number of broblasts in
the treatment group, while on the fourteenth
day there was a decrease in the number of
broblasts in the treatment group compared to
the control group (Table II). This is important
in wound healing, because in the early stages
of wound healing, broblasts play an important
role in stimulating proliferation, migration, cell
differentiation and controlling protein synthesis
and degradation [2]. However, at resolution
of the healing phase, the number of fibroblast
cells will decrease due to cell apoptosis [17].
In the treatment group, an increase in the number
of broblasts compared to the control group will
accelerate the healing process. This is evidenced
by a greater decrease in the treatment group on
the fourteenth day, which indicates the resolution
of the healing phase which proceeds earlier than
in the control group. Combination FD-PRP and HA
group had the best histopathological test results in
the form of the number of broblast cells compared
to other groups.
Based on the immunohistochemical review
of this study, it was found that the expression
of PDGF-BB was higher in the treatment group
compared to the control group on the third
day (Figure 1). Increased expression of growth
factors such as PDGF-BB can accelerate wound
healing. Verma et al. [18] who stated that the
application of autologous PRP can accelerate
soft tissue healing and bone regeneration
by increasing PDGF-BB. Lynch et al. [19]
stated that increasing PDGF in the wound area
can increase cell density in connective tissue.
In addition, there is the formation of thicker
connective tissue during the healing process. In
addition, Komatsu et al. [20] stated that PDGF-BB
enhances periodontal ligament regeneration
by decreasing osteocalcin cells and increasing
the proliferation and migration of fibroblast
cells periodontium. Nevins et al. [21] stated
that PDGF-BB can provide clinically signicant
attachment enhancement in the treatment of
moderate to severe two and three-walled infrabony
periodontal defects. Anusaksathien et al. [22]
stated that maintaining PDGF levels in the
third week after healing can inhibit growth
and biomineralization. This indicates that
continuous administration of PDGF can slow
cementoblast-induced mineral formation.
Based on the result of this study, it was
found that the BOP value in the treatment group
was lower than the control group, both on the
third day and on the fourteenth day (Table IV).
Table IV - Differences of BoP on days 3 and 14.
Group
Bleeding on probing (BoP)
Day 3 Day 14
x ± SD p value x ± SD p value
Control (-) 1,000 ± 0.000
0.348
a
1,000 ± 0.000
0.007
b
*
Control (+) 1,000 ± 0.000 1,000 ± 0.000
AH 0.670 ± 0.577 0.000 ± 0.000
FD-PRP 0.330 ± 0.577 0.000 ± 0.000
AH + FD-PRP 0.670 ± 0.577 0.000 ± 0.000
*significance p <0.05.
a
Kruskal Wallis test on 5 groups on day 3.
b
Kruskal Wallis test on the 5 groups on the 14
th
day.
7
Braz Dent Sci 2023 Apr/Jun;26 (2): e3680
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination of hyaluronic acid after periodontal surgical healing: an experimental study on animal model
Wulandari P et al.
Effectiveness freeze dried platelet rich plasma combination
of hyaluronic acid after periodontal surgical healing:
an experimental study on animal model
On the fourteenth day, all treatment groups had a
BOP value of 0, which indicated an inammation-free
surgical area. Abdul Ameer et al. [23] stated
that the application of PRP in the periodontal
pocket has an anti-inflammatory effect that
can accelerate the healing process. This will be
seen in the improvement of periodontal clinical
parameters, such as a decrease in plaque index,
gingival index, BOP and clinical attachment
level. Vera et al. [24] stated that hyaluronic acid
can have an indirect effect on inflammation,
stabilize granulation tissue and prevent the
release of protease enzymes from inammatory
cells that cause inammation can break down
extracellular matrix proteins during the healing
process. This research has limitations, including
the need to nd the best dose selection in the use
of freeze-dried platelet-rich plasma to support the
stability and durability of the material combined
with FDPRP and hyaluronic acid.
CONCLUSION
The combination of FD-PRP and HA has
accelerated the healing of periodontal surgical
wounds.
Acknowledgements
We gratefully thank the Research Centre
Universitas Sumatera Utara through Hibah Talenta
“Hibah Penelitian Dasar” Research Grant 2021.
Author’s Contributions
PW, DS, MA, B, RS: Researcher, Critically
Revised the Manuscript. PW: Writing, Analysis,
Interpretation. PW, DS, MA: Conception and
Data Design. B, RS: Performed the Experiments.
Conict of Interest
The authors certify that they have no
commercial or associative interest representing
a conflict of interest in connection with the
manuscript.
Funding
The authors are grateful for the research
funding provided by the Research Centre
Universitas Sumatera Utara through Hibah Talenta
“Hibah Penelitian Dasar” Research Grant 2021.
Regulatory Statement
Before the commencement of research, the
ethical approval for the current study was obtained
from the institutional ethical committee. The approval
code for this study is No. 0350/KEPH-FMIPA/2021.
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Pitu Wulandari
(Corresponding address)
Universitas Sumatera Utara, Faculty of Dentistry,
Department of Periodontics, Medan, Indonesia.
Email: pitu.wulandari@usu.ac.id
Date submitted: 2022 Nov 11
Accept submission: 2023 Apr 14
