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.e3366
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
Efeito da combinação de xenoenxerto e PRP na expressão de metaloproteinase de matriz -13 (MMP-13) durante regeneração óssea
Eka Fitria AUGUSTINA
1
, Nur Riianty RIVAI
2
, Haykal Miftah GIFARY
2
1 - Universitas Airlangga, Faculty of Dental Medicine, Department of Periodontology. Surabaya, Indonesia.
2 - Universitas Airlangga, Faculty of Dental Medicine. Surabaya, Indonesia.
How to cite: Augustina EF, Rivai NR, Gifary HM. The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in
bone regeneration. Braz Dent Sci. 2023; 26(2): e3366. https://doi.org/10.4322/bds.2023.e3366
ABSTRACT
Objective: inammation may play a role in bone loss by altering the bone remodelling process, favouring bone
resorption by osteoclasts over bone synthesis by osteoblasts. Matrix metalloproteinase 13 (MMP-13) has the ability
to activate osteoclasts, leading to bone resorption. Regenerative treatments have been widely used in periodontology.
When combined with Platelet-rich brin (PRF), xenografts will give better results in bone regeneration. The aim of
this study was to evaluate the effect of xenograft combined with PRF on MMP-13 expression in a bone defect using
an experimentally created bone defect. Material and Methods: eighteen New Zealand rabbits were assigned to three
groups. Each group consisted of six New Zealand rabbits. A critical bone defect with a diameter size of 5 mm was
created in the right tibia of each rabbit in group 1 (application: xenograft), group 2 (application: PRF), and group 3
(application: xenograft and PRF). The PRF was produced from 5 ml of blood taken from each rabbit’s ears. After 30 days,
the rabbits were euthanized. The tissue samples were evaluated by immunohistochemical staining. Results: group 3
showed the lowest mean expression of MMP-13 (4.50) compared to group 1 (20.50) and group 2 (11.70). Group 3
showed a signicant difference in the MMP-13 expression compared to group 1 and group 2 (P = 0.000) (P < 0.05).
Conclusion: this research showed that the combination of xenograft and PRF had the lowest expression of MMP-13.
The application of a xenograft and PRF has better osteogenesis ability in bone regeneration.
KEYWORDS
MMP-13; Xenograft; Platelet-rich brin; Bone regeneration; Inammation.
RESUMO
Objetivo: inamação pode interferir na perda óssea através de alterações no processo de remodelação, favorecendo
a reabsorção óssea pelos osteoclastos ao invés da síntese pelos osteoblastos. A metaloproteinases de matriz 13
(MMP-13) ativa osteoclastos causando reabsorção óssea. Tratamentos regenerativos têm sido amplamente usados
na periodontia. Quando combinamos Plasma rico em plaquetas (PRP) e xenoenxerto levam a melhores resultados de
regeneração óssea. O objetivo deste estudo foi avaliar os efeitos de xenoenxerto combinado com PRP na expressão de
MMP-13 em defeitos ósseos experimentais. Material e Métodos: dezoitos coelhos Nova Zelândia foram distribuídos
em 3 grupos de 6 coelhos cada. Um defeito ósseo de 5 mm de diâmetro foi feito na tíbia direita dos animais do grupo
1 (xenoenxerto), grupo 2 (PRP) e grupo 3 (Xenoenxerto+PRP). O PRP foi obtido pela coleta de 5mL de sangue das
orelhas dos coelhos. Após 30 dias, os coelhos foram eutanasiados. As amostras foram submetidas a coloração imuno-
histoquímica. Resultados: o grupo 3 apresentou a menor expressão de MMP-13 (4.50) quando comparado ao grupo 1
(20.50) e ao grupo 2 (11.70). O grupo 3 mostrou diferença estatística signicante em relação a expressão de MMP-13
quando comparado aos grupos 1 e 2 (p=0.000) (p< 0.05). Conclusão: esta pesquisa mostra que a combinação de
xenoenxerto e PRP teve a menor expressão de MMP-13. A combinação de xenoenxerto e PRP têm maior habilidade
de osteogênese na regeneração óssea.
PALAVRAS-CHAVE
MMP-13; Xenoenxerto; Plasma rico em plaquetas; Regeneração óssea; Inamação.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
INTRODUCTION
Bone defects due to infectious and other
pathological processes can lead to inammatory
processes, such as the bone destruction caused by
periodontal disease. Proinammatory mediators,
such as Interleukin-1 (IL-1), Interleukin-6 (IL-6)
and tumour necrosis factor α (TNFα), trigger the
expression of matrix metalloproteinases (MMPs).
MMPs actually play major roles in normal tissue
remodelling processes, such as in embryonic
development, bone growth, resorption and
wound healing [1]. MMPs also cause pathological
tissue destruction [2].
MMP-13 is a coupling factor that
periosteoclastic cells (osteoblast/bone lining
cells/osteoblast precursors/early reversal cells)
produce to aid the breakdown of the osteoid
prior to the attachment and resorption of
osteoclasts [3]. MMP-13 produced by osteoclasts
and polymorphonuclear (PMN) causes collagen
degradation in the connective tissue and alveolar
bone and affects the remodelling and degradation
of the periodontium [1]. MMP-13 can trigger the
activation of osteoclasts and is also expressed in
osteoblastic cells adjacent to osteoclasts at sites
of active bone resorption [2], gingival crevicular
epithelium, gingival broblasts, macrophages and
plasma cells [4]. MMP-13 affects the activity of
osteoclasts and bone resorption, thus contributing
to the destruction of periodontal tissue [5].
Bone graft materials are used to support
bone healing and are often used in guided tissue
regeneration (GTR) [6]. Bone grafts must be
biocompatible, bioresorbable and adequately
porous to be suitable for vascular ingrowth [7],
and they must also be suitable for osteoconduction,
osteoinduction and osteogenesis for bone tissue
regeneration. The addition of a bone graft is
expected to accelerate bone regeneration [8].
Xenografts are bone graft materials that are
derived from a genetically unrelated species to
the host [9]. New bone formation is slower with
xenografts than with allografts [10]. Xenografts
on bone defects can cause the differentiation of
mesenchymal cells and reduce cytokine levels and
control inammatory response [11].
Xenografts have been widely used in tissue
regeneration, but in clinical use, many studies
report that the use of xenografts alone leads to
less than optimal healing and tissue repair [12].
Research shows that xenografts give better
results when combined with the use of membrane
materials or other growth factors [13]. Xenografts
are known to have a long absorption capacity [14].
As shown in many studies, a xenograft remains
in the tissue nine months after application [3].
Therefore, xenografts are used in combination
with materials that can improve the regenerative
properties of the xenograft [15].
PRF is simple to produce, does not use
chemicals and produces growth factors such as
platelet-derived, vascular endothelial and insulin-
like growth factors. The literature shows that
PRF can continuously increase the proliferation
of all types of cells, especially osteoblasts [16].
These growth factors can regenerate bone tissue
and release growth factors continuously for up to
ten days [17]. PRF is also an immune regulation
node with inammation retrocontrol ability [14].
Several studies have reported that PRF
can support hard or soft tissue regeneration.
However, combining xenograft and PRF has never
been proven to decrease MMP-13 expression
in bone defect inammation. This study aimed
to evaluate the effect of xenograft and PRF on
MMP-13 expression in a bone defect using an
experimentally created bone defect in rabbits.
MATERIALS AND METHODS
Animal selection and study design
This study was an experimental in vivo study
using New Zealand rabbits. The rabbits selected
were eight-to-ten-week-old males, each weighing
1.5-2.5 kg. The rabbits were kept in 60 x 60 x
60 cm cages in isolation rooms with good air
ventilation. A normal chow diet and water were
provided ad libitum [18]. This study received
ethical approval from the Ethical Committee of
Dental Research, Airlangga University, reference
number 184/KKEPK.FKG/XII/2021.
The samples were chosen at random and
were determined using the Hosmer–Lemeshow
test sample formula. Eighteen rabbits were
assigned to three groups. Each group consisted of
six rabbits. A critical bone defect with a diameter
size of 5 mm was created in the right tibia of
each rabbit. In group 1, the defect was grafted by
xenograft (bovine, Indonesia) 0.05 gr; in group 2,
the defect was grafted by PRF. PRF was produced
from 5 ml of blood taken from each rabbit’s ears.
In group 3, the defect was grafted by xenograft
and PRF.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
PRF
The PRF was produced from 5 ml of blood
taken from the ears of each rabbit. The blood was
put in a glass tube and centrifuged for ten minutes
at 2500 rpm to form the PRF. Whole blood was
centrifuged to separate it into an upper liquid
layer of platelet poor plasma, a middle gel-layer
and a red blood cell (RBC) layer, with the PRF
being isolated by cutting the upper section of the
RBC. The PRF was placed in a lter, then cut into
small pieces and mixed with the xenograft using
a spatula [17,19].
Experimental procedure
Group 1 was grafted by xenograft. First,
intramuscular anaesthesia with ketamine
15–25 mg/kg (Anesject, Indonesia) was injected
into the right tibia. A lengthwise incision was
made in the right tibia. A separation of the bone
was performed, and a defect with a diameter size
of 5 mm and a depth of 3 mm was created using
a micromotor bur. The weight of each xenograft
(bovine bone, Indonesia) was 0.05 gr in every
defect in the right tibia [20]. The wound was
then stitched with inner and outer layer stitching
using silk thread. The wound was closed using
medical plaster (Hypax, Indonesia). After two
weeks, the outer layer was removed and left for
30 days [21].
In group 2, 5 ml of blood from each rabbit’s
ears was taken to make the PRF. The PRF
weight was 0.05 gr in every defect of the right
tibia. The same procedure was performed as for
group 1. The wait was 30 days.
In group 3, 5 ml of blood was taken from each
rabbit’s ears to make the PRF. The PRF weight
was 0.025 gr, and the xenograft (bovine bone,
Indonesia) weight was 0.025 gr in each defect of
the right tibia. The same procedure was performed
as for group 1. The wait was 30 days (Figure 1).
Tissue isolation
Intramuscular anaesthesia with ketamine
15–25 mg/kg (Anesject, Indonesia) was injected
into the right tibia. Tissue was taken from the
right tibia and put in a 10% neutral buffered
formalin xation solution. The xation was done
in two stages. After the rst 48 hours, the xation
solution was replaced, and the tissue was cut
smaller to aid penetration. At this stage, the tissue
was left in the solution for another 48 hours.
Observation of the expression of MMP-13 was
completed using the immunohistochemical
technique. First, the slides were washed with
phosphate buffered saline (PBS) pH 7.4 once
Figure 1 - The experimental procedure on the animal: bone defect creation in the right tibia (A), application of xenograft to the bone defect
(B), application of PRF to the bone defect (C).
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
for five minutes. Then, blocking endogenous
peroxide 3% H
2
O
2
was used for 20 minutes,
and then the slides were washed with PBS pH
7.4 three times for ve minutes.
After that, the unspecic protein was blocked
using 5% fetal bovine serum containing 0.25%
Triton X-100, and then the slides were washed
with PBS pH 7.4 three times for five minutes.
Then, the slides were incubated in a monoclonal
antiMMP-13 (Santa Cruz) 1:1000 dilution for
60 minutes and washed with PBS pH 7.4 three times
for ve minutes. Then, the slides were incubated
with antirabbit HRP (Biogear), conjugated for
40 minutes and washed with PBS pH 7.4 three
times for five minutes. Then, the slides were
dripped with diaminobenzidine, incubated for ten
minutes and washed with PBS pH 7.4 three times
for ve minutes and H
2
0 for ve minutes.
Counterstaining was completed using
Mayer’s Hematoxylin Solution, which was
incubated for ten minutes and then washed with
tap water. The slides were rinsed with dH
2
O
and dried. Mounting was completed using an
Entellan cover with cover glass. The samples were
observed under a light microscope.
Measuring MMP-13 expression
A total of 18 slides were divided into
three examination groups, with six slides per
group. Each tissue sample was cut into slices
with a thickness of 4 μm. Then, a haematoxylin
eosin (HE) examination was performed; the
osteoblast structure was observed, and an
immunohistochemical examination of MMP-
13 expression was completed. The examination
was performed using random numbering.
The examination and measuring of
MMP-13 expression were performed by observing
the brown colour of the cytoplasm cell, which had
been modied for osteoblastic cells [20,22]. Each
slide used 1000 × magnication and 20 elds of
view to measure the average.
The number of osteoblasts and osteoclasts
were observed using HE. The results were
calculated under a light microscope with a 1000 ×
magnication, each containing 1500 cells, then
the HE was used for structural comparison.
Statistical analysis
The expression of MMP-13 was analysed
using the Kolmogorov–Smirnov normality test.
Then, the result was tested by a one-way analysis
of variance to determine any differences between
groups and by a post hoc test, Tukey’s honestly
signicant difference test, with a signicance
value of p < 0.05.
RESULT
Quantity of osteoblasts and osteoclasts
The osteoclasts were observed and compared
with the osteoblasts. As shown in Figure 2, the
highest number of osteoclasts was in group 1 (15),
then group 2 (11) and group 3 (9). The highest
number of osteoblastic cells was in group 3 (19),
then group 1 (4) and group 2 (14).
MMP-13 expression
MMP-13 expression in the wound areas is
presented in Figure 3. Group 3 showed the lowest
mean expression of MMP-13 (4.50) compared
with group 1 (20.50) and group 2 (11.70)
(Figure 2). In group 3, there was a difference
signicant in MMP-13 expression compared with
group 1 and group 2 (P = 0.000) (P < 0.05).
DISCUSSION
Based on the immunohistochemical
examination, the mean expression of MMP-13 in
group 1 was 20.50 (Figure 2). Xenografts are
Figure 2 - The number of osteoblasts and osteoclasts in the bone
defect using HE examination (A), the mean of expression of MMP-13
in the bone defect (B).
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
known to have a long absorption capacity [23] and
remain in the tissue nine months after having been
applied [12]. Histologically, new bone formation
was lower than by allograft. The high expression
of MMP-13 in group 1, as demonstrated by
research, showed that in xenograft-treated bone,
bone remodelling is accompanied by chronic
inammation at a low level [24].
When chronic inflammation occurs,
inammatory mediators such as IL1 and TNFα
increase [10]. The Increased of IL-1 and TNFα will
stimulate increased of MMP-13 expression [4],
where MMP-13 or the collagenase-3 enzyme has a
vital role in bone biology [25]. MMP-13 degrades
not only type II collagen but also types I, III and
X, which are essential components of bone [4].
IL-1 and TNFα receptors, through ligand binding,
will recruit receptors that bind to proteins and
continue the stimulus into the cell. To bind
to the receptor, IL-1, and TNFα through a
phosphorylation mechanism in cells, mediated
by one of the proteins, namely mitogen-activated
protein kinase (MAPK). MAPK facilitates the
induction of MMP-13 due to the presence of
these inammatory mediators [7]. Inammatory
mediators present in these tissues cause increased
MMP-13 expression. This is consistent with the
theory that the high expression of MMP-13 in
gingival crevicular uid in periodontitis patients
suggests a role for increased MMP-13 in the bone
resorption process [4].
In group 2 (addition of PRF), the mean
number of MMP-13 expression was lower
(11.17) than in group 1. It can be seen that PRF
has anti-inammatory properties by releasing
inammatory mediators slowly; the number of
inammatory mediators is controlled, so the MMP-
13 expression also decreases. Physiologically, the
brin matrix of PRF can retain several growth
factors and inammatory mediators and release
them slowly to the defected area. Leukocytes
and inflammatory mediators, such as IL1β,
IL6, IL4 and TNF, are retained in the PRF, thus
providing an anti-inammatory effect [26].
The lowest MMP-13 expression, 4.50, was
found in group 3 (xenograft and PRF treatment).
PRF has properties that improve wound healing,
increase bone regeneration, stabilise graft
position and hemostasis [27]. With controlled
inammation, inammatory mediators decrease,
and the expression of MMP-13 also decreases,
increasing bone formation and the number of
osteoblasts. PRF is combined with a xenograft;
although it does not have an osteoinductive ability,
the inorganic material of this graft can make
Figure 3 - The immunohistochemical analysis of expression of MMP-13. The application of xenograft (A), PRF (B), xenograft and PRF (C). The
analysis of expression was completed in a light microscope with a magnification of 1000 × in 20 different fields.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3366
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration
Augustina EF et al.
The expression of matrix metalloproteinase-13 (MMP-13) on
xenograft and PRF in bone regeneration
an attachment and proliferate the osteoblastic
cells [28]. According to research by Simon (2012),
when combined with the use of membrane
material, GTR or other growth factors, a xenograft
gives a better result than when used alone [13].
HE staining showed a higher number of
osteoblasts in group 2 and group 3 than in
group 1. The formation of osteoblasts in the tibial
bone defect of rabbits treated with xenograft,
PRF and xenograft and PRF shows the process of
bone regeneration. This is because PRF contains
concentrations of growth factors and other
mediators that can improve wound healing and
increase bone regeneration. This can be seen from
clinical indicators, which include reduced pocket
depth, better attachment (an increased clinical
attachment level) and a more lled infrabony
defect area [29].
The tibia bone of the New Zealand rabbit
was used as a sample because it has a similar
bone anatomy and basic morphology to humans.
It was hoped that the results of this study would
demonstrate that the regenerative results of
adding regenerative materials to the defect show
a microscopic correlation between the formation
of osteoblastic cells in the rabbit tibia bone and
the human alveolar bone [30].
Some limitations need to be considered in
the interpretation of the results. First, further
research is needed to determine the cellular
mechanism of PRF for the tissue healing process,
especially in bone defects in dentistry. In addition,
research is needed to determine the effect of using
PRF and other regenerative materials as tissue
regeneration therapy materials, especially in the
eld of dentistry.
CONCLUSION
The results of this research showed that
the combination of xenograft and PRF had the
lowest expression of MMP-13. The application of
xenograft and PRF has better osteogenesis ability
in bone regeneration.
Author’s Contributions
EFA: Conduct research, data analysis, create
the script. NRR: Conduct research, data analysis,
create the script. HMG: Conduct research, data
analysis, create the script.
Conict of Interest
The authors have no proprietary, nancial or
other personal interest of any nature or kind in
any product, service and/or company presented
in this article.
Funding
This research did not receive any specic
grant from funding agencies in the public,
commercial or not-for-prot sectors.
Regulatory Statement
This study received ethical approval from the
Ethical Committee of Dental Research, Airlangga
University, reference number 184/KKEPK.FKG/
XII/2021.
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Eka Fitria Augustina
(Corresponding address)
Universitas Airlangga, Faculty of Dental Medicine, Department of Periodontology,
Surabaya, Indonesia.
Email: ekatri91@gmail.com
Date submitted: 2021 Dec 31
Accept submission: 2022 Nov 11
