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.e3847
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Potential effect of platelet rich fibrin prepared under different
centrifugation protocols on stem cells from the apical papilla
O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células
tronco da papila apical
Lina Samir SHALABY
1,2
, Sahar SHAWKAT
2
, Iman FATHY
3
1 - Newgiza University, School of Dentistry, Oral Biology Department, Giza, Egypt.
2 - Cairo University, Faculty of Dentistry, Oral Biology Department, Cairo, Egypt.
3 - Ain Shams University, Faculty of Dentistry, Oral Biology Department, Cairo, Egypt.
How to cite: Shalaby LS, Shawkat S, Fathy I. Potential effect of platelet rich brin prepared under different centrifugation protocols on
stem cells from the apical papilla. Braz Dent Sci. 2023;26(4):e3847. https://doi.org/10.4322/bds.2023.e3847
ABSTRACT
Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem
cells from the apical papilla (SCAP) seeded along with platelet rich brin (PRF) scaffolds prepared under two
different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used.
Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF)
groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S
(ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL)
immunouorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:
Both types of platelet rich brin increased the cell count, cell viability with no cytotoxicity that was reected on
increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed
signicant increase in proliferation and differentiation potentials compared to L-PRF group.
KEYWORDS
Alkaline phosphatase; Centrifugation; Platelet rich brin; RANKL ligand; Stem cells.
RESUMO
Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila
cultivadas conjuntamente com brina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação
distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram
divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem
de células e ensaio de viabilidade foram realizados para vericar a capacidade proliferativa. Coloração
vermelho de alizarina S, atividade de fosfatase alcalina e imunouorescência para o receptor ativador do fator
nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular.
Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que
reetiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo
A-PRF aumentou signicativamente a proliferação e diferenciação comparado com o grupo L-PRF.
PALAVRAS-CHAVE
Células-tronco; Centrifugação; Fibrina rica em plaquetas; Fosfatase alcalina; RANKL.
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INTRODUCTION
Dental trauma and caries both of which are
prevalent dental problems that leads to pulp
exposure, infection and necrosis. Under these
conditions, endodontic treatment is the most
common clinical treatment through which the
dental pulp is removed. In pediatric dentistry
and endodontics, the management of immature
permanent teeth remains a challenge. As once
the tooth lost its vitality, the root development
halts, leaving behind a weak tooth that unable to
withstand the normal physiological masticatory
forces. The consequences will be a high rate
of root fracture with poor prognosis in the
medium to the long term. Most of the studies
revealed that, teeth were lost in the rst 10 years
following trauma, despite being endodontically
treated in more than 50% of cases [1]. Tissue
engineering has been a topic of extensive
research over the past decade, involves a triad
(stem cells, scaffold and growth factors), aiming
to form a new tissue to restore the anatomy
and function as the original one. Regenerative
endodontic techniques (RETs) have been
recently introduced with the ultimate goal
of stimulating further root development and
thickening of root dentinal walls [2]. Stem-cell
biology has become an important eld for the
understanding of tissue regeneration. A variety
of dental MSCs have been isolated including stem
cells from bone marrow (BMSCs), dental pulp
(DPSCs), exfoliated deciduous teeth (SHED),
periodontal ligament stem cells (PDLSCs),
dental follicle precursor cells (DFPCs), stem cells
from apical papilla (SCAP) and gingiva- derived
mesenchymal stem cells (GMSCs). SCAP
are particularly relevant and significant in
regenerative endodontic procedures since they
are the cells suggested to populate the root
canal area following regenerative endodontics.
Hence, they should be targeted for maximum
benet of stem cell research and translational
medicine [3].
Platelets, isolated from a peripheral blood,
showed the ability of concentrated platelets to
provide 6–8 times supraphysiological doses of
growth factors. Earlier studies, demonstrated
the ability of several key growth factors, found
in platelets, to stimulate the recruitment and
differentiation of mesenchymal stem cells and
other target cells which markedly support tissue
regeneration [4]. The Platelet rich plasma
(PRP) was introduced to the world of dentistry
in 1997 by Whitman and co-workers. It was
suggested that PRP can attract stem cells from
surrounding periapical tissues. PRP was referred
as a first-generation platelet concentrate,
followed by the platelet rich brin (PRF) as a
second-generation platelet concentrate that was
developed rst by Choukroun et al. in 2001 and
the third- generation, called concentrated growth
factors (CGF) that was developed and described
in 2006 by Sacco [5].
Since PRF introduction in 2001, various
protocols utilizing the low-speed centrifugation
concept for PRF preparation, such as advanced
platelet rich fibrin (A-PRF) and injectable
platelet rich brin (i-PRF), have been proposed
with different amounts of growth factors
and other biomolecules necessary for tissue
regeneration and wound healing. The alteration
in centrifugation parameters, such as speed
and time, was showed to have a direct impact
on growth factors release within the PRF
matrix [4,5]. However, reference data about
potential effect of centrifugation parameters
modication on PRF matrix and its impact on
tissues regeneration still not properly covered
and needs further research. Hence, the present
work was designed to evaluate the proliferation
and differentiation potential of the stem cells
from apical papilla seeded along with platelet
rich brin scaffolds prepared under two different
centrifugation protocols which are standard and
advanced/ Low speed centrifugation concept
(LSCC) protocols.
MATERIALS AND METHODS
Stem cells isolation, characterization and culture
This study was approved by the research
ethics committees Faculty of Dentistry Cairo
University, number 19515. All experiments were
performed in accordance with the committee
guidelines of the stem cells experiment.
The current study was performed by using
human sound impacted third molars (n=12)
collected from healthy young patients
(18 to 21 years old) with incompletely formed
roots. The extracted teeth were immediately
rinsed with sterile PBS (PH 7.4) and transferred
in transfer solution (PBS + 10000 U penicillin/
streptomycin + preservative media) until being
transferred to the laboratory for further work.
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Patient recruitment
The current study was performed by
using human sound impacted third molars
(n=12) collected from healthy young patients
(18 to 21 years old) in the Oral and Maxillofacial
Surgery Department, Ain Shams University.
▪ Patient’s inclusion criteria were:
- Age ranges from 18 to 21 years old
- Sex, males and females
- Medically t and well
- Has the capacity to give an informed
consent
- Partially sound or fully impacted wisdom
teeth with incompletely formed roots
▪ Patient’s exclusion criteria were:
- Radiographic examination showed
completely formed roots
- Carious wisdom teeth
- Strategic tooth or not causing harm
during or upon its eruption
▪ Patients were informed about the nature of
the study, and they were asked to sign an
informed consent.
▪ The blood sample was collected later from
another adult healthy volunteer with normal
blood picture.
Isolation of SCAP
Apical papilla (soft tissue loosely attached
to the apices of incompletely formed roots) was
detached with a pair of forceps. Tissue obtained
from apical papillae of all teeth were mixed all
together. SCAP were isolated from this tissue
using enzymatic digestion method.
Characterization of SCAP
Characterization was done using flow
cytometric analysis. The immunoassaying
stains [CD45-PC5 (Phycoerythrin Cynin),
CD44-FITC (uorescein Isothiocyanate) and
CD73-PE (phycoerythrin)] were used to label
the isolated cells.
Culture Protocol of SCAP
The cells were cultured in T-75 culture
ask, in complete culture media (Dulbecco’s
modified Eagle’s medium (DMEM) (Gibco,
Invitrogen) containing 10% fetal bovine serum
(FBS) (Gibco) and 1% penicillin/streptomycin
(Gibco). Flask was incubated at 37 °C in an
atmosphere of 5% CO2. The media was changed
every 24 hours. When the cells reached 80%
confluency, the cells were harvested and
passaged. Cells from the 3rd passage were used
in the following assays.
PREPARATION TECHNIQUES OF PRF
A sample of blood was collected from an adult
healthy volunteer with normal blood picture in a
plain glass tube “without anticoagulants”, then
immediately centrifuged at room temperature
(20-25ºC) to prepare the PRF gel according to the
selected protocols in the current study design [6]
as follows:
▪ Standard Leucocytes PRF (L-PRF), sterile
plain glass-based vacuum tubes (10 ml;
2700 rpm for 12 minutes).
▪ Advanced PRF (A-PRF), sterile plain
glass-based vacuum tubes (10 ml; 1500
rpm for 14 minutes).
SEEDING OF THE SCAP AND GROUPING:
Seeding of the SCAP in the different groups
was done according to the study design for
7 days for osteogenic differentiation [7,8], into
4 different groups as follows;
1. Negative control (NC): SCAP + conventional
culture media*
2. Positive control (PC): SCAP + osteogenic
culture media (OM)**
3. Leukocyte PRF (L-PRF): SCAP + OM + L-PRF
4. Advanced PRF (A-PRF): SCAP + OM + A-PRF
* Conventional culture media: (Dulbecco’s
modied Eagle’s medium (DMEM) (Gibco,
Invitrogen) containing 10% fetal bovine
serum (FBS) (Gibco) and 1% penicillin/
streptomycin (Gibco).
** Osteogenic culture media (OM): low
glucose DMEM containing 10% fetal
bovine serum (FBS) and 1% penicillin/
streptomycin, 50ng/ml Ascorbic acid
and 10mM β-Glycerophosphate (Gibco,
Thermosientic, Germany).
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ASSESSMENT METHODS
Proliferation assessment
Cell counting ‘Trypan blue’ by Hemocytometer
The cells were counted by automated
hemocytometer to estimate the total number
of cells according to the following protocol [7].
The samples were loaded as suspension on
hemocytometer and observed. Nonviable cells
were stained; however, the viable cells did not
take up the stain.
Assessment of cell viability by cell cytotoxicity and
proliferation assay (MTT)
MTT assay was used to monitor the response
and health of cells in culture. The optical density
was assayed by spectrophotometer. The cell
cytotoxicity assay was performed according to
manufacturer instructions using the Vybrant®
Methyl-tetra-zolium (MTT; 3-(4, 5 dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide) Cell
Proliferation Assay Kit, cat no: M6494 (Thermo
Fisher, Germany).
Differentiation assessment
Assessment of inorganic deposition using Alizarin
red S (ARS) stain
ARS stain was used to assess the deposition of
inorganic content in differentiated SCAP, according
to the following protocol [9]. The microscopic
examination was performed by LABOMED
microscope with suitable magnication, cat no:
9126000; USA. Then the area fraction percentage
was calculated. The staining intensity was scored
according to a fourtier system: 0, no staining; 1+,
weak; 2+, moderate; and 3+, strong. In brief,
the H-score of each sample was calculated as
the sum of each intensity (0-3) multiplied by the
percentage of positive cells (0-100%). The score
ranged from 0-300. The median value of H-score
was calculated.
Assessment of Alkaline phosphatase (ALP) activity
The Alkaline phosphatase activity was
measured according to manufacturer instructions
in supernatant of differentiated SCAP using an
ALP assay kit (Sigma)® with para-nitrophenyl
phosphate (p-NPP) as substrate. 100 µL of
each p-nitrophenol standard and 50 µL of
each test sample was added to a 96-well plate.
After incubation at 37 ºC the absorbance was
measured immediately at 405 nm using a on a
spectrophotometer using an ELx800 absorbance
microplate reader (ELx 800; Bio-Tek Instruments
Inc., Winooski, VT, USA). A standard curve of
absorbance versus concentration was generated
and used to determine the ALP activity (U/L).
Assessment of RANKL expression in SCAP using
immunouorescence staining
Cells from different groups were harvested
and cultured for 24 hours on cover slips and
examined for the expression of Receptor activator
of nuclear factor kappa-Β ligand (RANKL) for
SCAP using specic polyclonal antibody [10].
The microscopic examination was performed
by LABOMED Fluorescence microscope with
suitable magnication, cat no: 9126000; USA.
The immunouorescence (IF) staining intensity
was scored according to a fourtier system: 0,
no staining; 1+, weak; 2+, moderate; and 3+,
strong. In brief, the H-score of each sample
was calculated as the sum of each intensity
(0-3) multiplied by the percentage of positive
cells (0-100%). The score ranged from 0-300.
The median value of H-score was calculated.
STATISTICAL ANALYSIS METHOD
All experiments were performed in triplicate.
All assays were repeated three times to ensure
reproducibility. Data were analysed using the
GraphPad prism version 9.3.1. (San Diego, US)
and used also for graph plotting. Each value
represents the mean ± standard deviation (SD).
Statistical significance was determined using
one-way analysis of variance (ANOVA) followed
by multiple comparison Tukey’s post-hoc test
to explore differences between multiple groups
means while controlling the experiment-wise
error rate. P-value: level of significance,
a p-value ≥ 0.05: means statistically insignicant,
p-value < 0.05 mean statistically significant,
p-value < 0.01: high statistically signicant.
RESULTS
Cell characterization (Flow cytometry)
Characterization of the isolated cells via
immunoassaying with stem cell markers CD44,
CD73 versus CD45 using ow cytometry revealed
that most of the cells showed double bright
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surface expression of CD44/CD73 in contrast
to only few of the cells were double negative
for both biomarkers. In order to confirm the
non- hematopoietic source of stem cells, the
CD73 and CD44 cells were gated with CD45
(hematopoietic stem cell marker). The obtained
results revealed that the CD44 and CD73 positive
cells, didn’t express CD45, which conrm that
the isolated stem cells were isolated from
non-hematopoietic source (Figure 1).
Cell counting (‘Trypan blue’ stain) by
hemocytometer
After 7 days, cell counting via trypan
blue showed negatively stained rounded
cells indicating viable cells, while positively
stained indicating dead cells (Figure 2). A-PRF
group showed the highest mean viable cell
count (4.27E+07) followed by L-PRF group
with mean viable cell count (7.32E+06).
Figure 1 - Characterization of cells using Multiparametric analysis: a representative FCM dot plots showing the gate protocol for cells. The cells
were stained with stem cell markers (CD73, CD44 and CD45). The CD44 and CD73 positive cells were gated in corresponding to CD45.
Figure 2 - A photomicrograph showing cell counting using trypan blue stain. Negative cells for Trypan blue were rounded indicating cell
viability in the experimental groups; positive stained cells indicated dead cells in the control groups. A: NC group, B: PC group, C: L-PRF group,
D: A-PRF group (Trypan blue stain, X100).
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Meanwhile the NC group showed mean viable cell
count (6.12E+05) higher than the PC group
(1.37E+05). Statistical analysis among the
L-PRF group and both NC and PC groups showed
that the viable cell count increase were not
signicant (P >0.05). While the viable cell count
increase among the A-PRF group and both NC
and PC groups were highly signicant (P< 0.01).
In addition to the viable cell count was higher in
A-PRF group when compared to L-PRF group and
this increase between the two test groups (A-PRF
and L-PRF) were statistically signicant (P< 0.05)
(Figure 3).
Cell viability and cytotoxicity (MTT assay)
After 7 days, the cell viability and cytotoxicity
in the 4 different groups using MTT assay. According
to optical density (OD) measured at 570 nm, A-PRF
group showed the highest viability levels (254.6%)
followed by L-PRF group (188.5%) and PC group
(157%) consecutively. However, the least levels
of OD were observed in the NC (99.6%) group.
Suggesting highest levels of proliferation on A-PRF
group followed by L-PRF group as compared to PC
and NC groups. Statistical analysis of cell viability
and cytotoxicity among the A-PRF group and both
NC (P < 0.0001) and PC (P < 0.001) groups were
found to be statistically signicant (P<0.05). The
cell viability was differed in P-value among the
L-PRF and both NC (P< 0.0001) and PC (P< 0.01)
groups. When results of the A-PRF group compared
to the L-PRF group, it was found to be statistically
signicant (P < 0.05) ((Figure 4).
Inorganic content deposition Alizarin red S
(ARS) stain
After 7 days of osteogenic differentiation,
cultured SCAP were stained with ARS stain to
identify nodules of calcication. No colorimetric
changes were detected in response to Alizarin
red stain in negative control group. Scattered
red stained nodules were observed in both
experimental and positive control groups. The
cells seeded on A-PRF showed the highest levels
of Average area fraction percentage of (92%)
followed by those seeded on L-PRF (68%) as
compared to PC (55%) and NC (0%), denoting
no formation of inorganic material. Nodules
were observed to be apparently increased in
size and intensity in A-PRF group as compared
to L-PRF and PC consecutively (Figure 5). The
morphometric analysis of the ARS stain was
statistically analysed among the 4 different groups.
Figure 3 - A graph showing cell count using Trypan blue stain
via hemocytometer of SCAP in the studied groups. Statistical
analysis was performed by one-way ANOVA followed by Tukey’s
post-hoc test, with the criterion for statistical significance as
follows:
* significant at P < 0.05, ** significant at P < 0.01, and ns
no significance.
Figure 4 - A graph showing cell viability assessment (MTT assay)
of SCAP in the studied groups. Statistical analysis was performed
by one-way ANOVA followed by Tukey’s post-hoc test, with the
criterion for statistical significance as follows: * significant at
P < 0.05, ** significant at P < 0.01, *** significant at P < 0.001 and
**** significant at P < 0.0001.
The average percentage of the area fraction of the
positively stained surface area was found to be
statistically signicant among the L-PRF group and
both NC (P< 0.0001) and PC (P < 0.01) groups.
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Figure 5 - A photomicrograph showing osteogenic differentiation of the SCAP in the studied groups. A. NC group, B. PC group, C. L-PRF group,
D. A-PRF group (Alizarin red S stain, X400).
Figure 6 - A graph showing alizarin red stain mineralized surface
areas of the SCAP in the studied groups. Statistical analysis was
performed by one-way ANOVA followed by Tukey’s post-hoc test,
with the criterion for statistical significance as follows: * significant
at P < 0.05, ** significant at P < 0.01, *** significant at P < 0.001 and
**** significant at P < 0.0001.
It was also signicant among the A-PRF group
and both NC (P < 0.0001) and PC (P < 0.0001)
groups. A highly significant difference was
noticed between the L-PRF and the A-PRF groups
(P < 0.0001) (Figure 6).
Assessment of alkaline phosphatase activity
(ALP assay)
After 7days, the alkaline phosphatase activity
was measured in the supernatant of differentiated
SCAP. The SCAP seeded on A-PRF showed the
highest levels of ALP secretion (155.8 U/L)
followed by those seeded on L-PRF (98.5 U/L) as
compared to PC (85.6 U/L) and NC (53.2 U/L).
ALP assay results were found to be statistically
signicant among all groups. With difference
in the signicance level among the groups as
follow; L-PRF group with NC (P< 0.0001) and
PC (P< 0.001) groups. A-PRF group with NC
group (P < 0.0001) and PC group (P < 0.0001).
And finally, L-PRF group with A-PRF group
(P < 0.0001) (Figure 7).
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RANKL protein expression
After 7 days, The SCAP seeded on A-PRF
group showed the highest levels of RANKL protein
expression (92%) followed by those seeded on
L-PRF group (78%) as compared to PC (72%)
and NC (35%) groups (Figure 8). RANKL protein
expression showed a statistically signicant results
among all groups as follow; L-PRF group with NC
group (P< 0.0001) and PC group (P< 0.0001).
A-PRF group with NC group (P < 0.0001) and PC
group (P < 0.0001). And nally, L-PRF group with
A-PRF group (P < 0.0001) (Figure 9).
DISCUSSION
In the last years, improvements were
accomplished in research and clinical levels of dental
pulp regeneration. Variable strategies in regenerative
endodontics have been proposed utilizing different
types of stem cells, scaffolds, and growth factors.
The success of regenerative endodontic therapy
(RET) depends on the regeneration triad key
elements (stem cells, scaffold, and growth factors).
Figure 7 - A graph showing Alkaline phosphatase assay of the
SCAP in the studied groups. Statistical analysis was performed by
one-way ANOVA followed by Tukey’s post-hoc test, with the criterion
for statistical significance as follows: * significant at P < 0.05,
** significant at P < 0.01, *** significant at P < 0.001 and **** significant
at P < 0.0001
Figure 8 - A photomicrograph showing immunofluorescence imaging of SCAP osteogenic differentiation in the studied groups. A. NC group,
B. PC group, C. L-PRF group, D. A-PRF group (anti-RANKL antibody, X400).
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PRF is an organized brin gel entrapping
platelets, immune cells, and leukocytes along
with releasing various growth factors and
cytokines [16]. Since its introduction in 2001
by Choukroun and co-workers, there has
been in-depth research regarding its clinical
applications and biologic actions. Various ways
were emerged to improve PRF characteristics
through preparation techniques modications
and optimizations. Besides the standard
Leukocyte PRF (L-PRF) protocol, researchers
introduced various PRF forms with greater
biological properties like advanced platelet-rich
fibrin (A-PRF) and injectable PRF (i-PRF).
This was obtained through using the LSCC.
This concept simply applied modifications in
centrifugation parameters (centrifugation time,
speed and g-force) to produce the PRF [5].
Since, the evidence stated that protocols with
reduction of the centrifugation force (g-force)
like A-PRF allow a greater cell count, better
distribution of cells of interest along with increase
in growth factors release, that reflected on
improvement in tissue regeneration process [5,6].
Moreover, Masuki et al., 2016 [16], reported
the A-PRF to have superior levels of platelets
and platelet-derived growth factors (TGF-β1,
PDGF-BB, VEGF), when compared to other PRP
preparations that was reected on enhanced cell
proliferation.
In the current study, PRF was prepared
according to two different previously published
protocols. The blood from a healthy volunteer
was collected in sterile glass tubes without
anticoagulants and centrifugated to obtain the
PRF gel. The first was the standard protocol,
L-PRF (10 ml; 2700 rpm for 12 minutes) and the
second was low speed concept protocol, A-PRF
(10 ml; 1500 rpm for 14 minutes). PRF Scaffolds
were immediately obtained then minced at
1x1 cm, in order to standardize its size [6].
In order to assess cell proliferation capacity.
Trypan blue stain and MTT assay were used to
evaluate the cell count and cell viability as well
as cytotoxicity, respectively. Both types of PRF
were found to promote the proliferation of SCAP
in vitro when compared to PC and NC groups, this
was in accordance with Hong et al., 2018 [8].
As according to Masuki et al., 2016 [16], PRF
has a slow and sustained release of key growth
factors for at least one week, meaning that
the PRF membrane stimulates its environment
for a significant time during remodelling.
Figure 9 - A graph showing RANKL protein expression of the SCAP
in the studied groups. Statistical analysis was performed by one-way
ANOVA followed by Tukey’s post-hoc test, with the criterion for
statistical significance as follows: **** significant at P < 0.0001.
Stem cells of apical papilla (SCAP) were the
cells of choice in the current study as they play
essential roles in the development and formation
of the tooth root [11]. The apical papilla is a stem
cell niche, that believed to provide odontoblasts
during tooth development. Moreover, SCAP were
reported to proliferate twice or triple times more
than DPSCs. In addition to showing the potential
to regenerate into vascularized dentin/pulp like
complexes in vivo [11,12]. Therefore, SCAP are
supposed to be a valuable stem cell source involved
in RET. It is readily available, conveniently
obtained, often-waste bound stem cells that lie in
each and every oral cavity at a certain moment in
time [3]. As, SCAP originate from a developing
tissue, such distinctive origin refers to the presence
of a percentage of early stem cells that could
empower special features in comparison to stem
cells derived from other more mature tissues [13].
PRF was the scaffold of choice in the current
study as it was recommended to be used by the
American Association of Endodontists (AAE) in
RET procedures. PRF is superior to PRP due to its
ease and inexpensive method of preparation and
lack of anticoagulants use [14]. It is collected in
a glass tube, not a plastic one as [15], considered
the silica of the glass tube to be a natural
coagulation inducer.
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This comes in accordance with our results which
showed better proliferative effects of PRF groups
over the control groups, with A-PRF superior to
L-PRF. Similar results were also reported with
other types of mesenchymal stem cells, including
human dental pulp cells [12] and periodontal
ligament stem cells [17].
The potential mechanism of the proliferation-
promoting effect may be due to the abundant
growth factors released from L-PRF and A-PRF.
In the current study, A-PRF was assumed to
remarkably stimulate the proliferation of SCAP
more than L-PRF. This could be explained according
to Masuki et al. [16], who considered the A-PRF
not only as a scaffold but also as a reservoir for
certain growth factors delivery at the site of
application. So, when A-PRF degrades, growth
factors are released to the media and stimulate
cell proliferation According to the literature, it was
known that A-PRF had a higher concentration of
growth factors within its brin matrix, increasing
the tissue regeneration rate when applied in
a surgical wound [6]. This fact allied to the
higher traction average and maximal traction of
A-PRF that make it more suitable material for
regeneration over the L-PRF. Pascoal et al. [18],
observed that LSCC provided a high mechanically
resistant membrane, besides the more even
distribution of cells throughout the brin clot. This
indicates that, when the A-PRF applied in multiple
situations, it may be more effective.
In the current study, regarding the cell
viability and proliferative capacity of SCAP
seeded on L-PRF scaffold, no signicant statistical
differences were shown between the L-PRF and
control groups after 7 days culture period. This
was the same as reported by Huang et al. [12],
who found that no significant effect in trypan
blue uptake and cell count in DPSCs treated with
or without PRF after 5 days of culture, with no
cytotoxic effect. In addition, Khurana et al. [7],
reported that, the viability of cells (DPSCs and
PDLSCs) cultured with PRF was statistically
insignicant when compared to cells cultured with
culture media (control group). Moreover, the PRF
membrane as a scaffold exhibited no cytotoxic
effects on DPCSs or PDLSCs. On the other hand,
Chang et al. [19], suggested that PRF increased
osteoblast proliferation in a time-dependent
manner. This can be explained as PRF may
have better influence on differentiated active
cells (osteoblast) as compared to its effect on
undifferentiated cells (SCAP, DPSCs and PDLSCs).
In general, PRF maintained viability of cells
without inducing apoptosis, as it has a potent
mitogenic activity [20]. This was evidently
proven through the current study. Where the
cell count and MTT assay results of both A-PRF
& L-PRF were found to significantly increase
the cell count and enhance the viability and the
proliferative capacity of the SCAP when compared
to the control groups after 7 days incubation
period (P < 0.05). This was in agreement with
Zhao et al. [17], who observed the number of
PDLSCs in the groups with the PRF membrane
were signicantly higher than that in the control
group throughout the seven days incubation
period. As for the MTT assay, the absorbance
values for the growth curves for all the groups
increased during the testing period, and there was
a signicant difference between PRF-containing
groups and the control group (without PRF). Also,
A-PRF group exhibited a superior effect on the
proliferation rate than L-PRF group.
In the current study, after conrmation of
the biocompatibility and the positive effect of
both types of PRF on SCAP proliferation, the
differentiation capacity was assessed using ARS
stain, ALP assay and RANKL protein expression. In
order to identify L-PRF & A-PRF biological effects
on osteogenic differentiation, alizarin red stain was
used to verify the state of osteogenic differentiation
in terms of extracellular matrix mineralization
deposition [9]. The average percentage of the area
fraction of the positively ARS-stained surface area
was statistically signicant among the L-PRF group
and both NC and PC groups. It was also signicant
in the A-PRF group and both NC and PC groups.
As well as a highly signicant difference between
the L-PRF and the A-PRF groups. These observation
of the major mineralization events after 7 days of
culture in the PRF groups were in accordance with
previous reports where the PRF was assumed to
increase the mineralized nodules in mesenchymal
stem cells [21], suggesting similar osteogenic
potentials that was completely not evident in the
negative control group.
Alkaline phosphatase is the main early
osteogenic differentiation marker. In the current
study, after seven days of incubation, the SCAP
seeded on A-PRF group showed the highest
levels of ALP activity followed by those seeded
on L-PRF group as compared to PC and NC
groups. ALP assay results were statistically
signicant among all the groups. With difference
in the significance level among the groups.
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Different centrifugation protocols of PRF on SCAP
Shalaby et al.
Different centrifugation protocols of PRF on SCAP
Which was relevant to several studies that showed
PRF to increase the activity of alkaline phosphatase
in cells of the mesenchymal lineage isolated from
dental pulp [12], periodontal ligament [21] and
apical papilla [8]. Conversely, one study carried
out by Zhao et al. [17], showed an inhibitory
effect of PRF on alkaline phosphatase activity and
subsequently the osteoblastic differentiation of
PDLSCs. This could be explained due to the different
type of cells (PDLSCs) and the PRF preparation
protocol (10-mL of blood centrifuged at 400 g for
10 min) utilized in their study. Furthermore, two
other studies reported the PRF failure to change
alkaline phosphatase activity, as it did not change
alkaline phosphatase activity in in rat calvaria
osteoblasts [22] and bone marrow cells [23].
Regarding the difference in the two previously
mentioned studies, the conflict may be clarified due
to the different type of stem cells and centrifugation
parameters utilized. In the first study, the authors
used the stem cells from animal source (rat calvaria
osteoblasts) and the PRF preparation protocol was
different (400 g for 10 min). And in the second
study that could be clearly explained as the authors
treated PDLSCs with PRF subjected to different
centrifugation parameters. As they utilized Low
(700 rpm for 3 minutes) and Medium (1300 rpm
for 8 minutes) relative centrifugation force (RCF),
in addition to the use of a plastic not a glass tubes
for blood collection and centrifugation. Taken all
together, all studies reported an increase of alkaline
phosphatase in response to PRF exposure, except for
those three studies. The study that reported PRF to
inhibit ALP secrtion and the other two studies that
showed no effect of PRF on ALP secretion.
Receptor activator of nuclear factor-kappa
B (NF-κB) ligand (RANKL) is a membrane-
associated cytokine. Its expression is induced
preferentially in immature cells. The expression
of RANKL has been linked to the differentiation
state of osteoblastic cells. In the current study
RANKL protein expression was detected and
showed statistically signicant results among all
groups, suggesting that both L-PRF and A-PRF
groups signicantly enhanced the RANKL protein
expression compared to the control groups,
indicating the osteoblastic differentiation state of
the present SCAP, seeded on both PRF scaffolds.
This could be due to the released growth factors
within the PRF matrix such as platelet derived
growth factor (PDGF), insulin growth factor-1
(IGF-1) and Transforming growth factor-β (TGF-β)
that plays an important role in osteogenesis.
The PDGF, provokes proliferation, migration and
survival of stem cell lineages [24]. Since each
platelet contains around 1,200 molecules of PDGF,
abundant concentration of PDGF in PRF may
lead to more profound effect on wound healing
and bone regeneration. Where IGF-1 induces
differentiation and mitogenesis of mesenchymal
cells, in addition to stimulation of chemotaxis and
activation of osteoblasts resulting in bone formation.
Moreover, TGF-β enhances osteoblast proliferation
and deposition, together with the inhibition of
osteoclasts formation and bone degeneration [4].
This was clearly expressed and supported in ARS
stain and ALP assay present results that indicated
increased mineralization and differentiation of SCAP
into osteoblast like cells. This was previously clearly
explained by Ikebuchi et al. [25], who revealed that
RANKL-RANK signaling regulates osteoblastogenesis
in addition to its role in osteoclastogenesis.
As maturing osteoclasts secrete vesicular RANK
which activates RANKL reverse signaling in
osteoblasts and promotes osteoblast differentiation.
During osteoblastogenesis, the RANK expression is
reduced and RANKL forward signaling on osteoblast
differentiation is relieved [26]. The current results
were also, in accordance with You et al. [27], who
found that PRF has a signicant effect on bone
regeneration as it accelerated the mineralization in
the vicinity of the osteoblast cell line. In addition
to upregulation of the biomarker genes such as
collagen type I, BMP-2, and osteocalcin, which are
associated with bone formation. The ndings of
Sumida et al. [28], suggested that PRF enhanced
early stage osteogenesis through expression of
osteoblastic differentiation makers, including
BMP 2 and 4 (bone morphogenic proteins -2 & 4)
and RUNX2 (Runt-related transcription factor 2).
In addition to optimizing osteoblastic differentiation,
as PRF increased the OPG/RANKL ratio by
inducing OPG expression, with no effect on the
expression of RANKL. Our results were opposed
to Chang et al. [19], who found that RANKL
expression was not signicantly (p > 0.05) altered
by PRF while increased the osteoprotegerin (OPG)
secretion. The authors claimed that the positive
effect of PRF on proliferation of osteoblasts/
stromal cells was due to the signicant increase
in osteoprotegerin (OPG) secretion, while the
insignicant expression of RANKL that has a role
in osteoclastogenesis. And this conict could be
explained due to the different type of cells used
and the PRF preparation protocol they used in
their study, which is different from that used in the
current study.
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Shalaby et al.
Different centrifugation protocols of PRF on SCAP
Shalaby et al.
Different centrifugation protocols of PRF on SCAP
In the present work, the differentiation
assessment methods were used to evaluate the
osteogenic capacity of PRF compared to the
control groups, along with comparing the L-PRF
and A-PRF osteogenic potential. The A-PRF group
showed signicantly higher results in all these tests
compared to L-PRF group. Hence, A-PRF group is
assumed to have higher osteogenic proliferation and
differentiation capacity than L-PRF group. This was
in accordance with Fujioka-Kobayashi et al. [29],
who stated that the modications to centrifugation
speed and time with the low-speed concept
favour an increase in growth factor release from
PRF clots. This, in turn, may directly influence
tissue regeneration by increasing cell migration,
proliferation, and collagen mRNA levels.
The present results were also, in agreement
with Ansarizadeh et al. [30], who found that
A-PRF application may result in enhanced new
bone formation and may aid in accelerating bone
formation through enhancing osteoblast activity and
bone formation. Though it could be useful for bone
formation in clinical medicine. Pascoal et al. [18],
were the rst to suggest the superiority of A-PRF,
that may be due to its signicant higher maximal
traction score and average traction compared to
L-PRF. The mechanical properties of A-PRF in
specic the higher resistance to tensile strength than
L-PRF which consequently may inuence the time
for membrane degradation and the release of growth
factors. With further promotion of proliferation and
differentiation of the surrounding cells.
From the investigations and within the
limitation of the current study, the following were
concluded, PRF was proven to be a biocompatible
scaffold with no cytotoxic effect as it increased the
cell count and proliferative capacity of the SCAP
compared to the control groups (without PRF).
Both forms of PRF enhanced the SCAP osteogenic
differentiation potential through increased
deposition of mineralization nodules accompanied
with increased ALP activity and RANKL protein
expression levels when compared to the control
groups (without PRF). The LSCC which represented
in the form of A-PRF showed signicant increase
in the proliferative and differentiation capacity of
the SCAP compared to L-PRF.
Hence, both forms of PRF could be adopted in
regenerative endodontics with superior benecence
of A-PRF, especially when used in presence of
SCAP. Further animal and clinical studies are
needed to emphasize our ndings and to explore
the underlying molecular mechanisms.
Acknowledgements
This research was performed in Central lab
of stem cells and biomaterials applied research
(CLSCBAR)
Author’s Contributions
LS, IF: Conceptualization, Methodology,
Software, Validation, Formal Analysis,
Investigation, Resources, Data Curation, LS:
Writing – Original Draft Preparation, SS, IF:
Writing – Review & Editing, Visualization,
Supervision LS: Project Administration and
Funding Acquisition.
Conict of Interest
Authors declare no conict of interest.
Funding
This research received no external funding.
Regulatory Statement
This study was conducted in accordance with
all the provisions of the local human subjects
oversight committee guidelines and policies of:
Faculty of Dentistry Cairo University, Egypt. The
approval code for this study is: 19515.
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Different centrifugation protocols of PRF on SCAP
Shalaby et al.
Different centrifugation protocols of PRF on SCAP
Lina Samir Shalaby
(Corresponding address)
Newgiza University, School of Dentistry, Oral Biology Department, Giza, Egypt.
Email: lina-samir@dentistry.cu.edu.eg
Date submitted: 2023 Apr 02
Accept submission: 2023 Nov 10
