Reduction of relative centrifugal forces increases growth factor release within solid platelet-rich-fibrin (PRF)-based matrices: a proof of concept of LSCC (low speed centrifugation concept)

Abstract

Purpose The present study evaluated the platelet distribution pattern and growth factor release (VEGF, TGF-β1 and EGF) within three PRF (platelet-rich-fibrin) matrices (PRF, A-PRF and A-PRF+) that were prepared using different relative centrifugation forces (RCF) and centrifugation times. Materials and methods immunohistochemistry was conducted to assess the platelet distribution pattern within three PRF matrices. The growth factor release was measured over 10 days using ELISA. Results The VEGF protein content showed the highest release on day 7; A-PRF+ showed a significantly higher rate than A-PRF and PRF. The accumulated release on day 10 was significantly higher in A-PRF+ compared with A-PRF and PRF. TGF-β1 release in A-PRF and A-PRF+ showed significantly higher values on days 7 and 10 compared with PRF. EGF release revealed a maximum at 24 h in all groups. Toward the end of the study, A-PRF+ demonstrated significantly higher EGF release than PRF. The accumulated growth factor releases of TGF-β1 and EGF on day 10 were significantly higher in A-PRF+ and A-PRF than in PRF. Moreover, platelets were located homogenously throughout the matrix in the A-PRF and A-PRF+ groups, whereas platelets in PRF were primarily observed within the lower portion. Discussion the present results show an increase growthfactor release by decreased RCF. However, further studies must be conducted to examine the extent to which enhancing the amount and the rate of released growth factors influence wound healing and biomaterial-based tissue regeneration. Conclusion These outcomes accentuate the fact that with a reduction of RCF according to the previously LSCC (described low speed centrifugation concept), growth factor release can be increased in leukocytes and platelets within the solid PRF matrices.

Full text

Vol.:(0123456789)
1 3
Eur J Trauma Emerg Surg (2019) 45:467–479
DOI 10.1007/s00068-017-0785-7
ORIGINAL ARTICLE
Reduction ofrelative centrifugal forces increases growth factor
release withinsolid platelet-rich-fibrin (PRF)-based matrices:
aproof ofconcept ofLSCC (low speed centrifugation concept)
K.ElBagdadi1· A.Kubesch1· X.Yu2· S.Al-Maawi1· A.Orlowska1· A.Dias1·
P.Booms1· E.Dohle1· R.Sader1· C.J.Kirkpatrick1· J.Choukroun1,3· S.Ghanaati1
Received: 7 November 2016 / Accepted: 10 March 2017 / Published online: 21 March 2017
© The Author(s) 2017. This article is an open access publication
Moreover, platelets were located homogenously through-
out the matrix in the A-PRF and A-PRF+ groups, whereas
platelets in PRF were primarily observed within the lower
portion. Discussion the present results show an increase
growthfactor release by decreased RCF. However, further
studies must be conducted to examine the extent to which
enhancing the amount and the rate of released growth fac-
tors influence wound healing and biomaterial-based tissue
regeneration. Conclusion These outcomes accentuate the
fact that with a reduction of RCF according to the previ-
ously LSCC (described low speed centrifugation concept),
growth factor release can be increased in leukocytes and
platelets within the solid PRF matrices.
Keywords Inflammation· Leukocytes· Platelets·
Platelet-rich-fibrin· Tissue engineering· Vascularization
Introduction
Various blood concentrates are used to support tissue
regeneration and wound healing in different fields. One of
these systems is platelet-rich plasma (PRP), a technique
that has been developed for clinical practice and tissue
regeneration therapies [1, 2]. PRP is prepared by multiple
centrifugation steps using patient blood to which antico-
agulants have been added to achieve a platelet-rich concen-
trate that can be used for different indications [3]. However,
seeking to minimize contamination risk, eliminate addi-
tional anticoagulants and use the autologous and natural
regeneration capacity, a new system, platelet-rich fibrin
(PRF), was introduced as the first blood concentrate system
without additional anticoagulants [4].
PRF is derived from patient venous blood by means of
single-step centrifugation without the further addition of
Abstract Purpose The present study evaluated the plate-
let distribution pattern and growth factor release (VEGF,
TGF-β1 and EGF) within three PRF (platelet-rich-fibrin)
matrices (PRF, A-PRF and A-PRF+) that were prepared
using different relative centrifugation forces (RCF) and
centrifugation times. Materials and methods immunohis-
tochemistry was conducted to assess the platelet distribu-
tion pattern within three PRF matrices. The growth factor
release was measured over 10 days using ELISA. Results
The VEGF protein content showed the highest release on
day 7; A-PRF+ showed a significantly higher rate than
A-PRF and PRF. The accumulated release on day 10 was
significantly higher in A-PRF+ compared with A-PRF and
PRF. TGF-β1 release in A-PRF and A-PRF+ showed sig-
nificantly higher values on days 7 and 10 compared with
PRF. EGF release revealed a maximum at 24h in all groups.
Toward the end of the study, A-PRF+ demonstrated sig-
nificantly higher EGF release than PRF. The accumulated
growth factor releases of TGF-β1 and EGF on day 10 were
significantly higher in A-PRF+ and A-PRF than in PRF.
Electronic supplementary material The online version of this
article (doi:10.1007/s00068-017-0785-7) contains supplementary
material, which is available to authorized users.
* S. Ghanaati
shahram.ghanaati@kgu.de
1 FORM (Frankfurt Orofacial Regenerative Medicine) Lab,
Department forOral, Cranio-Maxillofacial andFacial Plastic
Surgery, University Hospital Frankfurt Goethe University,
Theodor-Stern-Kai 7, 60590FrankfurtamMain, Germany
2 Department ofOrthopedics, West China Hospital/West China
School ofMedicine, Sichuan University, Chengdu, Sichuan,
People’sRepublicofChina
3 Private Practice, Pain Therapy Center, Nice, France
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468 K.El Bagdadi et al.
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any type of anticoagulants. This system was developed
to fulfill clinical needs by being time-saving and easy to
use [4]. PRF-based matrices include various inflamma-
tory cells, such as platelets and leukocytes, in combination
with various plasma proteins embedded in a fibrin network
[5]. The components of PRF-based matrices are known to
play an important role during the process of wound heal-
ing. Platelets are the first cells to occur in the region of an
injury. In addition to their role within hemostasis, platelets
have inflammatory potential, including the recruitment of
further inflammatory cells, such as neutrophils and mac-
rophages, and promote angiogenesis and tissue repair [6,
7]. In this context, platelets are able to express a series of
biologically active signaling molecules and growth factors,
such as platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF) and transforming growth
factor beta (TGF-β). These growth factors are essential
for tissue vascularization and new tissue formation [8,
9]. Moreover, platelets contain granules with cytokines,
chemokines and other inflammatory mediators that are
released after platelet aggregation to enhance hemostasis
and activate and recruit cells to the site of inflammation
[10, 11]. Leukocytes also contribute to angiogenesis and
lymphangiogenesis by participating in cell–cell cross talk
and expressing various signaling molecules [12, 13]. The
extracellular matrix in the wound bed supports the forma-
tion of blood vessels, and fibrin provides a scaffold for the
inflammatory cells [14].
The structure and constituents of PRF-based matri-
ces were previously explored by our group. An ex vivo
histomorphometrical study showed a dense structure and
specific localization of the included inflammatory cells
in the lower part of PRF [5]. In addition, a modification
of the preparation setting based on the previously LSCC
(described low-speed centrifugation concept) is a first step
in the reduction of the applied relative centrifugation force
(RCF). This step was accompanied by a mild increase of
centrifugation time, resulting in a so-called advanced PRF
(A-PRF) [5, 15]. Analysis of the structure and composi-
tion of A-PRF revealed a more porous structure compared
to PRF [5]. In addition, histomorphometrical analysis
revealed significantly more neutrophilic granulocytes in the
group of A-PRF compared with PRF [5].
While developing PRF-based matrices, the focus was
on clot formation, consistency and functional integrity the
fibrin clot and the distribution of the included inflammatory
cells to generate PRF-based matrices with high functional-
ity and adequate handling. In this study, the applied RCF
and centrifugation times are key elements. Further research
on PRF-based matrices regarding their structure and com-
position indicates that adjusting the centrifugation time,
i.e., reducing the spinning time and applying the same RCF
as in the case of A-PRF, allows the introduction of a new
PRF-based matrix, Advanced-PRF+ (A-PRF+). A previ-
ous systematic study demonstrated the influence of the RCF
reduction on the leukocyte and platelet numbers as well as
their role in growth factor release in fluid PRF-based matri-
ces following the LSCC, which indicates that reducing the
RCF enhances the cell number and growth factor release
within PRF-based matrices [15]. Based on the LSCC, we
examined modifications of the RCF and centrifugation
times in solid PRF-based matricesand their influence on the
growth factor release within the previously introduced PRF
protocols with a solid structure; PRF, A-PRF and A-PRF+.
Therefore, the goal of the present study was to determine
growth factor release in solid PRF-based matrices, PRF,
A-PRF and A-PRF+, at six different time points over a
period of 10 days. Additionally, immunohistochemical
analysis was conducted to assess the platelet distribution
pattern within the various PRF-based matrices.
Materials andmethods
PRF preparation
For each protocol, peripheral blood was drawn from four
healthy volunteers between 25 and 60 years of age (two
females, two males) without a history of anticoagulant
usage. Informed consent was obtained from each donor
who participated in this study. As previously described
[5], the venous blood was collected in 10-ml sterile glass
tubes (A-PRF tubes Process for PRF™, Nice, France; Mec-
tron, Cologne, Germany) without external anticoagulants
and placed immediately in a centrifuge (Duo centrifuge,
Process for PRF™, Nice, France; Mectron, Cologne, Ger-
many). The centrifuge has a fixed angle rotor with a radius
of 110 mm and no brake. After centrifugation time, the
centrifugation process ends automatically, and the centri-
fuge stops in 2–5s. All preparation steps were performed
at room temperature according to the established protocols
as follows:
• PRF: 10ml; 2400rpm; 12min; 708g
• A-PRF: 10ml; 1300rpm; 14min; 208g
• A-PRF+: 10ml; 1300rpm; 8min; 208g
After centrifugation, all clots were carefully removed
from the tubes and separated from the red blood cell frac-
tion with sterile tweezers and scissors.
PRF cultivation
The total clots of PRF, A-PRF and A-PRF+ were placed
in separate wells of a 6-well plate (Greiner, Bio-One Inter-
national) and covered with 5 ml Roswell Park Memorial
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469
Reduction ofrelative centrifugal forces increases growth factor release withinsolid…
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Institute medium (RPMI 1640, Gibco Thermo Fischer
Scientific) without Fetal Bovine Serum and supplemented
with L-glutamine and 1% penicillin/streptomycin. The clots
were incubated in a humidified incubator for up to 10days
at 37 °C with 5% CO2. The supernatants from each well
were taken after 6, 24, 48, 72h, 7 and 10 days and stored
as aliquots at −80 °C. At each time point, all of the clots of
PRF-based matrices were placed into new wells and cov-
ered with 5ml fresh medium.
Growth factor measurement
The supernatants that were collected from the various
PRF-based matrices at different cultivation time points
were used for the quantification of different growth fac-
tors by enzyme-linked immunosorbent assay (ELISA). All
collected supernatants were simultaneously centrifuged
(1500rpm; 5min.) using a centrifuge (Thermo fisher sci-
entific, Heraeus® Labofuge® 400 R) to exclude possible
residue that could affect the photometrical measurement.
Before TGF-β1 and EGF ELISA preparation, the super-
natants were diluted 1:4 with the same cell culture RPMI
medium used for PRF-matrices cultivation. The protein
concentrations of human VEGF, TGF-β1 and EGF were
determined by the Dou Set ELISA kit (Human VEGF
DY293B, R&D Systems, detection range: 2000–31.3 pg/
ml), HumanDou Set ELISA kit (Human TGF-β1 DY240,
R&D Systems, detection range: 2000–31.3pg/ml) and the
Duo Set DuoSet ELISA kit (human EGF DY236, R&D
Systems, detection range: 3.91–250 pg/mL) according to
the manufacturer´s instructions. Measurements were con-
ducted using a microplate reader (Infinite® M200, Tecan,
Grödig, Austria) set to 450nm and subtracted at 570nm
from the 450nm measurements.
Immunohistological analysis
As previously described [5, 16], the PRF clots were col-
lected after 10 days and fixed in Roti®-Histofix 4%, acid
free (pH 7), and 4% phosphate-buffered formaldehyde
solution (Carl-Roth) for 24 h. The PRF-based matrices
were dehydrated in a series of alcohol and xylene through
a Tissue Processor (TP1020, Leica Biosystems Nussloch
GmbH, Germany) and embedded in paraffin blocks. After-
wards, 3µm thick sections from each sample were cut by
a rotatory microtome (Leica RM2255, Wetzlar, Germany).
For immunohistochemistry, the sections were deparaffi-
nized, rehydrated and finally sonicated in citrate buffer
(pH 6) at 96 °C for 20min. The sections were stained with
monoclonal mouse anti-human CD61 marker (1:50, Plate-
let Glycoprotein IIIa/APC, Clone Y2/5, Dako) by means of
an autostainer (Lab vision Autostainer 360, Thermo Fisher
Scientific). Histological examination was conducted using
a light microscope (Nikon Eclipse 80i, Tokyo, Japan).
Three of the authors KE, SA and SG, were independently
blinded for the morphological analysis. The micropho-
tographs were prepared with a connected DS-Fi1/Digi-
tal camera (Nikon, Tokyo, Japan) and a Digital sight unit
DS-L2 (Nikon, Tokyo, Japan).
Statistical evaluation
Data were expressed as the mean ± standard deviation.
Statistical analysis was conducted using Prism Version 6
(GraphPad Software Inc., La Jolla, USA). The significance
of differences among means of data was analyzed using
two-way analysis of variance (ANOVA) with the Tukey
multiple comparisons test (α = 0.05) of all pairs. The sig-
nificant differences were regarded as significant if the p val-
ues were less than 0.05 (*p < 0.05) and highly significant
if the p values were less than 0.005 (**p < 0.005), 0.0005
(***p < 0.0005) or 0.0001 (****p < 0.0001).
Results
General observation offibrin clotting withinthethree
investigated groups
Macroscopic observation demonstrated the formation
of three slightly different clots. PRF formed a clot with a
fibrin/red blood count (RBC) ratio of 1/1.66, and the clot
length was measured as 3.5cm. A-PRF showed a clot for-
mation with a fibrin/red blood count (RBC) ratio of 1/2.
Here the clot length was 3.5 cm. A-PRF+ had a fibrin/
red blood count (RBC) ratio of 1/3 and a length of 2.5cm
(Fig.1). Moreover, while separating the fibrin clot from the
RBC, it was observed that in the case of PRF and A-PRF,
the adhesion between the two sections, the fibrin clot and
RBC, was stronger compared with A-PRF+. Accordingly,
the A-PRF+ fibrin clot was much easier to separate.
Growth factor release kinetics fromtheclots
The present study focused on the determination of the
released growth factor kinetics of the three PRF-based
matrices, PRF, A-PRF and A-PRF+. The growth factors
VEGF, EGF and TGF-β1 were quantified for the released
concentrations at each time point (6, 24, 48, 72h, 7, and
10 days). Additionally, the accumulated growth factor
quantities were calculated.
VEGF release
The general trend of the three evaluated groups at each
time point was similar. The release of VEGF increased in
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470 K.El Bagdadi et al.
1 3
the very early phase from 6 to 24h in all groups. At 48h,
the growth factor release was comparable to the values at
24 h in all groups. From 48 to 72 h, a slight decrease in
the release of VEGF was evidenced in all groups. From
72 h to day 7, a highly significant increase in all groups
was observed (p < 0.0005) in an intra-individual com-
parison (data not shown). During the 4days of cultivation
between 72 h and day 7, the highest released concentra-
tion of VEGF over the study time was measured. Here,
A-PRF+ showed the highest concentration when com-
pared with PRF and A-PRF (PRF = 158.5 ± 36.6 pg/ml;
A-PRF = 153.6 ± 40.1 pg/ml; A-PRF+ = 242.35 ± 67.9 pg/
ml), which was statistically highly significant when com-
pared to PRF and A-PRF (p < 0.0005). By contrast, A-PRF
showed no statistically significant difference compared to
PRF. From day 7 to day 10, all groups showed a decrease in
the release of VEGF. This decrease was intra-individually
statistically highly significant compared with day 7 (data
not shown). Furthermore, after 10 days, A-PRF+ showed
the highest VEGF release (PRF = 83.7 ± 28.81 pg/ml;
A-PRF = 64.84 ± 15.7pg/ml; A-PRF+ = 95.5 ± 44.7pg/ml).
At this time point, no significant difference could be identi-
fied among the groups (Fig.2a1).
Concerning the accumulated VEGF concentra-
tion, a general trend was also evidenced by a continuous
increase in the released VEGF over the study time. In the
early phase (6–72 h), the release of VEGF increased in
all groups, whereas the groups’ concentrations were quite
similar. Moreover, in the late study period (72h–10days),
a similar tendency was observed in all groups. However,
A-PRF+ released the highest concentration on day 10 when
compared with PRF and A-PRF (Table1). This difference
was highly significant when comparing A-PRF+ to A-PRF
(***p < 0.0005) and significant comparing A-PRF+ to PRF
(**p < 0.005) at this time point (Fig.2 a2).
TGF-β1 release
Various TGF-β1 release patterns were measured in PRF,
A-PRF and A-PRF+. Within the PRF group, a slight
increase was observed in the early study time (6–72 h)
followed by a dramatic decrease in the late study time
(72h–10days). At 72 h, PRF already showed the highest
concentration over the study period. At this time point,
PRF was significantly higher only when compared to
A-PRF (p < 0.0001), whereas no significant difference was
observed compared to A-PRF+ (Fig.2b1).
The A-PRF group showed a high release value
at the first time point (6 h) (PRF = 4.6 ± 1.0 ng/ml;
A-PRF = 7.0 ± 1.4 ng/ml; A-PRF+ = 5.8 ± 1.4 ng/ml), the
difference between A-PRF and PRF being statistically sig-
nificant (p < 0.05). However, no statistically significant dif-
ference was detected regarding A-PRF+. This observation
was followed by irregular behavior until 72h and a signifi-
cant increase at day 7, when the highest TGF-β1 release of
A-PRF was observed. At this time point, A-PRF was sig-
nificantly higher than PRF (p < 0.0001), whereas no signifi-
cant difference was revealed for the A-PRF+ group.
A-PRF+ showed a mild decrease of the released TGF-
β1 at the early study time (6–48h). However, from 72h to
day 7, an increase in the released TGF-β1 was observed
when the highest concentration of TGF-β1 release was
reached in the case of A-PRF+. At day 7, a statistically
highly significant difference was observed when compared
with PRF (p < 0.0001), whereas no significant difference
was observable compared to A-PRF (PRF = 1.9 ± 1.6 ng/
ml; A-PRF = 8.5 ± 0.6 ng/ml; A-PRF+ = 8.6 ± 0.4 ng/ml).
From day 7 to day 10, the release of TGF-β1 decreased in
all groups. However, A-PRF showed significantly higher
values when compared with PRF (p < 0.0001). Similarly,
A-PRF+ revealed more growth factor release, which was
highly significant when compared with PRF (p < 0.0001).
No statistically significant difference was observed
when comparing A-PRF and A-PRF+ at this time point
(Fig.2b1).
The accumulated concentration of TGF-β1 showed
an increase in all groups at the early study time (6–72h).
However, at the late study time (72h–10days), the growth
factor release differed among the various groups. PRF
showed a more or less constant concentration of TGF-β1
after 72h, whereas in the case of A-PRF and A-PRF+, an
Fig. 1 The PRF-based matrices immediately following centrifuga-
tion
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471
Reduction ofrelative centrifugal forces increases growth factor release withinsolid…
1 3
Fig. 2 Statistical analysis of the growth factor releases by time
points as the mean ± standard deviation for PRF, A-PRF and
A-PRF+. a1 VEGF, b1 TGF-β1 release, c1 EGF release, (*p < 0.05),
(***p < 0.0005), (****p < 0.0001). Total accumulated growth factor
concentration over 10days. a2 VEGF, b2 TGF-β1, c2 EGF
Table 1 Accumulated growth factor concentration of PRF, A-PRF and A-PRF+ at day 10 as the mean ± standard deviation. Statistical analysis
of A-PRF and A-PRF+ compared with PRF (*p < 0.05), (**p < 0.005), (***p < 0.0005), (****p < 0.0001)
Growth factor PRF A-PRF A-PRF+
VEGF (pg/ml) 632.26 ± 90.58 593.15 ± 114.08 773.88 ± 117.66**
TGF β1 (ng/ml) 23.18 ± 1.22 34.081 ± 3.21**** 36.29 ± 5.73****
EGF (pg/ml) 858.62 ± 152.90 1106 ± 57.74* 1147.07 ± 164.47**
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472 K.El Bagdadi et al.
1 3
increased TGF-β1 concentration was observed. These dif-
ferences on day 10 were statistically significant when com-
paring A-PRF to PRF (p < 0.0001) and A-PRF+ to PRF
(p < 0.0001); however, no statistically significant difference
was detected when comparing A-PRF to A-PRF+ (Table1)
(Fig.2b2).
EGF release
A general trend was observed in all three PRF-based
matrices. The rate of the released EGF increased quite
early in the study time (6–24 h) to reach the highest
value in all groups at 24 h. At this time point, A-PRF+
showed the highest value of the released EGF when com-
pared with PRF and A-PRF (PRF = 282.69 ± 109.09 pg/
ml; A-PRF = 373.75 ± 101.25 pg/ml;
A-PRF+ = 435.17 ± 89.29 pg/ml), the difference being
statistically highly significant when comparing A-PRF+
to PRF (***p < 0.0005); no statistical significance was
observed when comparing A-PRF to A-PRF+. Subse-
quently, a course change was observed when a strong
reduction of the released EGF occurred in all examined
groups until 72h. After that, on day 7, a slight increase was
observed in all groups. Here also, A-PRF+ was the highest
(PRF = 148.28 ± 48.27 pg/ml; A-PRF = 138.70 ± 61.07 pg/
ml; A-PRF+ = 173.50 ± 98.72 pg/ml) although no statisti-
cally significant difference was detectable. At the last eval-
uated time point on day 10, all groups showed a significant
decrease in the released EGF compared with day 7 (data
not shown). However, at this time point, no statistically
significant differences were observed among the groups
(Fig.2c1).
The accumulated concentration of the released EGF
also exhibited a general trend. All groups showed a sim-
ilar curve progression in the form of increased EGF
release over the study time. A-PRF and A-PRF+ also dis-
played similar values. Early in the study time, a remark-
able increase in released EGF was evidenced in all groups.
After 72h, only a minor increase of the released EGF was
observed toward the end of the study on day 10. At these
time points (72 h–10 days), A-PRF and A-PRF+ showed
statistically significantly higher release values when com-
pared with PRF (A-PRF+ compared with PRF p < 0.005;
A-PRF compared with PRF p < 0.05), whereas no statisti-
cally significant differences were revealed when comparing
A-PRF to A-PRF+ (Table1) (Fig.2c2).
Platelet distribution inthePRF-based matrices
Immunohistochemical staining with CD-61 antibodies
against platelets was conducted to determine the platelet
distribution in cross sections of the three PRF-based matri-
ces. The platelet distribution was evaluated with regard to
the location in the clot. The platelets formed accumulations
within all three clots. PRF, which was prepared with a high
RCF, showed a different distribution pattern according to
the localization. The upper and middle portions of the clot
showed only a few platelets, whereas the majority of plate-
lets were distributed in the lower portion of PRF (Fig.3).
By contrast, A-PRF, which was prepared with a reduced
RCF, presented a different distribution pattern. Platelets
were dispersed all over the clot (Fig.4). A-PRF+ with a
reduced RCF and a reduced centrifugation time also dis-
played an even platelet distribution pattern in the various
locations within the clot (Fig.5).
Discussion
This study presents the potential of PRF-based matrices
(PRF, A-PRF and A-PRF+) for growth factor release as a
modest contribution to ongoing discussions regarding the
preparation of PRF-based matrices as biological scaffolds
and a natural growth factor release system, which is derived
from autologous blood. The results revealed continuous
growth factor release of VEGF, TGF-β1 and EGF over the
study time. However, statistically significant differences
among the various preparation protocols, PRF, A-PRF and
A-PRF+, were demonstrated.
One of the most potent angiogenesis-stimulating growth
factors is VEGF. A-PRF+ released significantly more
VEGF than PRF and A-PRF on day 7. Moreover, the
accumulated release of VEGF on day 10 was significantly
higher in A-PRF+ than in PRF and A-PRF. However,
no statistical significance was detected when evaluating
A-PRF and PRF. These outcomes are quite likely related to
the specific fibrin structure and cellular distribution pattern
of A-PRF+. VEGF plays a crucial role in wound healing
and tissue regeneration to promote vascularization and new
vessel formation [17]. Additionally, previous studies have
demonstrated that the sustained release of VEGF promotes
epithelialization and enhances collagen tissue deposition in
a skin wound healing model in mice [18]. Thus, the sus-
tained and enhanced VEGF release of A-PRF+ could lead
to more benefits in regeneration and vascularization and
thus provide a nutrient supply to support wound healing
and improve the biomaterial-guided regeneration pattern.
The release of TGF-β1 in A-PRF and A-PRF+ indi-
cated the maximal release values on days 7 and 10, which
were significantly higher when comparing A-PRF to
PRF and A-PRF+ to PRF. However, no statistically sig-
nificant difference between the TGF-β1 release of A-PRF
and A-PRF+ was identified. On day 10, the accumulated
TGF-β1 concentration was significantly higher in the
A-PRF and A-PRF+ groups than in the PRF group. By
contrast, A-PRF and A-PRF+ revealed no statistically
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473
Reduction ofrelative centrifugal forces increases growth factor release withinsolid…
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significant difference in this case. TGF-β 1 is essential
for wound healing [19]. Chronic wounds were observed
to have a decreased expression of TGF-β receptors [20].
Thus, PRF matrices with an enhanced release of TGF-
β1, as was the case for A-PRF and A-PRF+, could have a
major influence on wound healing as a catalyzer of wound
repair stages. In addition, this growth factor is known to
stimulate fibroblast migration, enhance collagen synthesis
and promote angiogenesis [21, 22]. All of the latter char-
acteristics are essential in the biomaterial-based regen-
eration process. Hence, PRF-based matrices as an addi-
tional autologous dose of inflammatory cells and growth
factor could be promising in the field of guided bone and
tissue regeneration (GTR and GBR), in which biomateri-
als should provide a scaffold and support the regeneration
process in the defect area.
Fig. 3 CD-61 immunohistochemical analysis of PRF according to
the different regions. a1, a2 upper portion; b1, b2 middle portion; c1,
c2 lower portion (a1, b1, c1 total scan sections; ×100 magnification,
scale bar 500µm). a2, b2, c2 show the distribution pattern of plate-
lets (yellow arrows) in higher magnification (f fibrin; b buffy coat;
×400 magnification; scale bar 20µm)
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474 K.El Bagdadi et al.
1 3
The release of EGF was generally higher in the A-PRF
and A-PRF+ groups when compared with PRF. Statisti-
cally highly significant differences were detected when
comparing A-PRF+ with PRF after 24h, whereas no sig-
nificant difference was observed between A-PRF+ and
A-PRF. The accumulated EGF release showed significantly
higher rates in the case of A-PRF and A-PRF+ compared
with PRF at most time points, particularly on day 10. EGF
has previously been described as promoting cell growth
[21], enhancing keratinocyte migration [23], inhibiting
apoptosis under hypoxic conditions [24], and supporting re-
epithelization and skin healing [25, 26]. Additionally, EGF
supports the healing process of chronic wounds [27], non-
healing chronic wounds and ulcers, which are, for example,
observed in diabetic patients known to lack the necessary
growth factors to maintain the healing process [28, 29].
Thus, such patient groups may benefit from the application
of PRF matrices as an autologous drug delivery system.
Fig. 4 CD-61 immunohistochemical analysis of A-PRF according to
the different regions. a1, a2 upper portion; b1, b2 middle portion; c1,
c2 lower portion (a1, b1, c1 total scan sections; ×100 magnification,
scale bar 500µm). a2, b2, c2 Show the distribution pattern of plate-
lets (yellow arrows) in higher magnification (f fibrin; b buffy coat;
×400 magnification; scale bar 20µm)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

475
Reduction ofrelative centrifugal forces increases growth factor release withinsolid…
1 3
Moreover, immunohistochemical evaluation indicated an
equal distribution pattern of platelets in all clot regions
in the case of A-PRF and A-PRF+, whereas in PRF, the
majority of the platelets were located in the lower portion
of the clot. These findings may be related to the LSCC (low
speed centrifugation concept), indicating that reducing
the applied RCF increases the number inflammatory cells
and platelets as well as the growth factor release within
the PRF-based matrices [15]. Because the centrifugation
process depends on cell weight and density, a higher RCF
may be the reason for the sedimentation of the majority
of the platelets to the lower portion of the clot according
to their density and size, as observed in PRF. Decreasing
the RCF allows the platelets to become separated from the
red blood cell phase and become equally distributed within
the fibrin network. The effectiveness of PRF clots with
low platelet counts and uneven platelet distribution may
have less influence on clinical outcomes than clots with
Fig. 5 CD-61 immunohistochemical analysis of A-PRF+ according
to the different regions. a1, a2 upper portion; b1, b2 middle portion;
c1, c2 lower portion (a1, b1, c1 total scan sections; ×100 magnifi-
cation, scale bar 500µm). a2, b2, c2 Show the distribution pattern
of platelets (yellow arrows) in higher magnification (f fibrin; b buffy
coat; ×400 magnification; scale bar 20µm)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

476 K.El Bagdadi et al.
1 3
evenly distributed and enhanced platelet numbers because
the applied clot could have uneven biological activity and
thus a reduced growth factor release, as indicated in the
present study. However, comparative clinical studies are
necessary to evaluate the advanced PRF matrices presented
here to demonstrate the extent to which the improved struc-
ture, even cellular distribution and enhanced growth factor
release may affect clinical outcomes.
These observations highlight the influence of RCF
reduction, i.e., from PRF (708 g) to A-PRF and A-PRF+
(208g) on platelet distribution, thereby correlating with the
previously demonstrated automated cell counting that indi-
cated significantly more platelets in PRF matrices prepared
with low RCF than with high RCF application [15]. A pre-
vious ex vivo immunohistochemical study demonstrated the
distribution pattern in PRF and A-PRF, which included, in
addition to platelets, a wide range of inflammatory cells
that physiologically exist within the peripheral blood, such
as leukocytes, including neutrophils and monocytes as well
as lymphocytes [5]. However, further immunohistochemi-
cal studies are required to determine the distribution pat-
tern of the included leukocytes and their subgroups, par-
ticularly in A-PRF+. These cells, particularly platelets and
neutrophilic granulocytes, contribute to neoangiogenesis
and VEGF release [30, 31]. In addition, platelets are the
primary secretory cells of EGF and TGF-β1 [32]; thus,
their presence within the PRF-based matrices is a possible
explanation for the observed growth factor release. These
cells are essential for wound healing and tissue regenera-
tion [33, 34]. In the present study, release kinetics displayed
an increased growth factor release over the study time and
a maximum at day 7 in the case of VEGF and TGF-β1 as
well as an increased growth factor release at 24h in the
case of EGF. Based on the growth factor and release kinet-
ics demonstrated here, one may assume that the growth fac-
tor release pattern within the various PRF-based matrices is
an active release from living cells within the different PRF
clots, which most likely experienced apoptosis during the
study period if 10days reflects the reduction in growth fac-
tor release at day 10 compared with day 7 in all groups and
growth factors.
Additionally, leukocytes and platelet interaction via
cellular cross talk have been described in bone regenera-
tion [9]. In this context, the high regeneration potential of
advanced PRF-based matrices could be beneficial in vari-
ous clinical applications, such as enhancing the regen-
eration pattern of biomaterials in terms of GTR and GBR.
Moreover, autologous biologizing biomaterials using PRF-
based matrices may improve the regeneration pattern in
large-sized, soft and bony defects to catalyze wound heal-
ing and regeneration. Ongoing clinical observations in oral-
and maxillofacial surgery have demonstrated that various
bony defects within the jaw or head can be regenerated by
different clot numbers according to the defect size. Thus,
molar sockets are treated with 2–3 clots, whereas larger
bony head defects are treated with up to 6 clots. Based on
these observations, PRF-based matrices could be a ben-
eficial tool to improve the regeneration of soft and bony
defects after orthopedic or trauma surgery. The present
study demonstrates that the application of the LSCC (low
speed centrifugation concept), by decreasing the RCF from
PRF toward A-PRF and A-PRF+, results in a significantly
higher release of VEGF, TGF-β1 and EGF. Notably, the
accumulated release over 10days of TGF-β1 and EGF sup-
ports the relation between the reduction of RCF and the
growth factor release. Hence, A-PRF+ and A-PRF, which
were prepared with the same RCF, displayed comparable
results that were significantly higher than PRF, which was
prepared with more than three times higher RCF. These
observations emphasize the fact that the application of the
LSCC is valuable in modifying and optimizing solid PRF-
based matrices. However, the manipulation of the centrifu-
gation time appeared to influence only certain growth fac-
tors, as shown in the case of A-PRF+. The accumulated
VEGF release on day 10 showed a significantly higher rate
in the group of A-PRF+ compared with A-PRF and PRF.
It may be that the application of a low RCF but a longer
centrifugation time, as demonstrated in the case of A-PRF,
affected the VEGF release capacity, whereas the applica-
tion of a low RCF and slightly decreased centrifugation
time, as in A-PRF+, resulted in a significantly higher
VEGF release. Another plausible explanation may be that
the specific fibrin clot composition of A-PRF+ allows a
highly increased VEGF release and thus a higher accumu-
lated VEGF release on day 10. These data accentuate the
fact that the various growth factor concentrations may be a
consequence of the various total cell concentrations within
the PRF-based matrices.
The various release profiles of the evaluated PRF-
based matrices may also be a consequence of the differ-
ent growth factor binding affinities to fibrin. It has been
demonstrated that growth factors, such as VEGF, have a
high affinity to bind to fibrinogen and fibrin so that those
factors are released in a sustained manner [35]. This
information is reflected in the present results by showing
significantly enhanced VEGF release on day 7 in the case
of A-PRF+. By contrast, EGF is released in a high con-
centration level at the very early time point of 24h. One
explanation for this observation may be the low binding
affinity of EGF to fibrin and fibrinogen [36]. Another
factor may be the structure of the PRF-based matrices.
A-PRF and A-PRF+ exhibit a more porous structure
than the densely structured PRF [5]. The physical prop-
erties of the clot and the specific fibrin structure related
to the manufacturing protocol [5] may also influence the
binding affinity and the sustained release of the various
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477
Reduction ofrelative centrifugal forces increases growth factor release withinsolid…
1 3
growth factors. It is possible that a more porous struc-
ture, as shown in A-PRF and A-PRF+, is one reason for
an enhanced growth factor release [5]. Thus, it remains
questionable whether the growth factor release is related
to the specific physical properties of the fibrin network or
to the included inflammatory cells and platelets, or per-
haps a combination of both. Therefore, further study is
required to understand this specific complex system.
The release kinetics of growth factors in the PRF-based
matrices have previously been reported in several studies
[37, 38]. Direct comparisons of these studies are limited
because of the various preparation protocols in terms of
RCF, centrifugation time, blood volume and the techniques
used to generate the PRF-based matrices. However, one
in vitro study analyzed the growth factor release in PRF-
based matrices compared with PRP [39]. Correlations
were demonstrated in the case of the accumulated TGF-β1
and EGF, for which both studies presented a significantly
higher growth factor release in PRF matrices prepared with
a low RCF application compared with PRF matrices with
high RCF exposure. This accentuates the fact that reduc-
tion of the RCF enhances the release of these growth fac-
tors. Notably, the later study also showed that PRP released
higher growth factor concentrations (EGF, VEGF and
TGF-β1) at the very early time points, whereas PRF-based
matrices showed a continuous and higher growth factor
concentration over a period of 10days [39]. Moreover, this
group demonstrated further evaluation of the growth fac-
tors in PRF, A-PRF and A-PRF+ [40]. The results of the
accumulated growth factor release on day 10 are consistent
with the present findings with regard to A-PRF+ concern-
ing TGF β1 and EGF. Both studies presented a significantly
higher release of these growth factors within A-PRF+
when compared with PRF. By contrast to Kobayashi etal.
(2016), the present study reveals no significant differ-
ences between A-PRF and A-PRF+ with regard to TGF
β1 and EGF. Additionally, the present outcomes indicate
significantly higher accumulated VEGF release on day 10
in the group of A-PRF+ compared with A-PRF and PRF,
whereas Kobayashi etal. (2016) showed no statistically sig-
nificant differences between the examined groups on day
10. At this point, it must be stressed that the two studies
were of different designs. Kobayashi etal. (2016) evaluated
different time points from the time points investigated in
the present study. In addition, Kobayashi etal. (2016) used
a shaking incubator before performing the ELISA evalua-
tion, whereas our group incubated the PRF-based matrices
without further manipulation, which can also be a reason
for the discrepancies revealed in the results. It is evident
that detection of the specific growth factors is dependent
on the specific methods employed. Thus, further studies in
this field are necessary to develop and evaluate PRF-based
matrices generated according to LSCC.
The present experimental design regarding the prepa-
ration and cultivation of PRF-based matrices may offer
advantages because the PRF clots were not compressed or
manipulated but nevertheless yielded the large amount of
growth factors in the PRF clot. Furthermore, the clots were
incubated in a cell culture environment to provide adequate
gas exchange and optimal conditions for cells. The pri-
mary limitation of this study is the invitro system issue. A
comparison with clinical results is difficult because of the
discrepancy of comparing the physiological environment
in vivo. Thus, the cellular crosstalk and enzymatic deg-
radation of the fibrin network would be different invivo.
Further invivo studies are required to determine the influ-
ence of the growth factors on the regeneration pattern of
PRF-based matrices, particularly those matrices that are
prepared according to the LSCC. This is necessary to iden-
tify out whether the observed inflammatory cell and growth
factor enhancement will contribute to an improved regen-
eration potential invivo. Moreover, the optimal release of
growth factors required in wound healing and regeneration
processes remains unclear, as is whether enhancing the
amount released will indeed lead to improved performance.
Thus, controlled clinical studies are essential to evaluate
the regeneration potential of A-PRF and A-PRF+ and to
establish the extent to which homogeneously distributed
platelets and an enhanced growth factor release in addition
to the porous structure will contribute to improved wound
healing.
Less is known regarding the interaction of the PRF-
based matrices with biomaterials with a view to improving
biomaterial-based regeneration. In addition, little atten-
tion has been focused on the composition of PRF-based
matrices obtained from patients undergoing pharmaco-
logic treatments and whether the growth factor release will
be influenced by medication. In addition, the regeneration
potential of the PRF-based matrices may also be related to
the age of the donor. Therefore, it may be that as the age
of donors increases, less growth factor is released and vice
versa. If this scenario is true, PRF-based matrices with
enhanced growth factor release may be beneficial in these
specific cases. In this respect, the determination of mono-
nuclear cell growth in PRF and penetration into the PRF-
based matrices as a simulation of the regeneration process
in vitro would be of interest in understanding the role of
PRF-based matrices in biomaterials and tissue engineering.
Hence, further studies of the PRF-based matrices as a com-
plex system that influences cell growth and differentiation
and provides a growth factor reservoir remain necessary.
Additionally, the current PRF-based matrices were pre-
pared according to specific protocols with a defined amount
of blood. However, it would be interesting to determine
how increasing or decreasing the blood volume influences
the composition of the prepared PRF-based matrices, their
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

478 K.El Bagdadi et al.
1 3
regenerative potential and their growth factor release. These
questions are current investigation topics of our research
group as we seek to enhance wound healing and tissue
regeneration to decrease patient morbidity. Hence, the out-
comes of this study could provide new clinical approaches
in tissue and bone regeneration in terms of a combination
of biomaterials with PRF-based matrices. Nevertheless,
further studies, particularly clinical studies, are required to
develop optimized, standardized and tailored preparation
protocols for various clinical applications and to demon-
strate their advantages now and in the future.
Conclusion
The present study demonstrates the influence of RCF
reduction on the growth factor release and platelet distribu-
tion in solid PRF-based matrices. A-PRF+, prepared with
a reduced RCF, displayed significantly higher VEGF con-
centration over the study period of 10days than A-PRF and
PRF, which exhibited no statistically significant difference.
EGF and TGF-β1 were comparable in A-PRF and A-PRF+,
which were significantly higher than PRF. Additionally, the
platelet distribution pattern appeared to be equivalent in
all regions concerning A-PRF and A-PRF+, whereas PRF
showed the largest accumulation of platelets in the lower
portion of the clot. Long-term, sustained and slow release
of growth factors from all of the PRF groups may support
cell migration and cell proliferation as well as offer advan-
tages in the wound healing process. However, the signifi-
cantly enhanced release in A-PRF and A-PRF+ may render
these matrices superior to PRF in specific clinical indica-
tions. These promising findings offer an excellent handling
efficiency and new approaches to the clinical application of
wound healing as well as soft and bone tissue regeneration.
Nevertheless, further clinical studies must demonstrate the
extent to which the application of LSCC to generate A-PRF
and A-PRF+ will benefit clinical outcomes.
Acknowledgements The authors thank the excellent technical sup-
port of Mrs. Verena Hoffmann.
Compliance with ethical standards
Conflict of interest Choukroun is the owner of PROCESS. The
authors declare no conflict of interest. The study was funded by the
FORM-lab.
Research involving human participants Blood samples of volun-
teers were used. Informed consent was obtained. No ethical approval
was required for this study.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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