CLINICAL

Advanced Platelet-Rich Fibrin: A New Concept for Cell-

Based Tissue Engineering by Means of Inflammatory Cells
Shahram Ghanaati, MD, DMD1,2*
Patrick Booms, PhD1
Anna Orlowska, BSc, DVM1
Alica Kubesch1,2
Jonas Lorenz, DDS1
Jim Rutkowski, DMD, PhD3
Constantin Landes, MD, DMD, PhD1
Robert Sader, MD, DDS, PhD1
CJ Kirkpatrick, MD, PhD, DSc2
Joseph Choukroun, MD1,4

Choukroun’s platelet-rich fibrin (PRF) is obtained from blood without adding anticoagulants. In this study,
protocols for standard platelet-rich fibrin (S-PRF) (2700 rpm, 12 minutes) and advanced platelet-rich fibrin (A-
PRF) (1500 rpm, 14 minutes) were compared to establish by histological cell detection and histomorphometrical
measurement of cell distribution the effects of the centrifugal force (speed and time) on the distribution of cells
relevant for wound healing and tissue regeneration. Immunohistochemistry for monocytes, T and B -
lymphocytes, neutrophilic granulocytes, CD34-positive stem cells, and platelets was performed on clots
produced from four different human donors. Platelets were detected throughout the clot in both groups,
although in the A-PRF group, more platelets were found in the distal part, away from the buffy coat (BC). T- and
B-lymphocytes, stem cells, and monocytes were detected in the surroundings of the BC in both groups.
Decreasing the rpm while increasing the centrifugation time in the A-PRF group gave an enhanced presence of
neutrophilic granulocytes in the distal part of the clot. In the S-PRF group, neutrophils were found mostly at the
red blood cell (RBC)-BC interface. Neutrophilic granulocytes contribute to monocyte differentiation into
macrophages. Accordingly, a higher presence of these cells might be able to influence the differentiation of host
macrophages and macrophages within the clot after implantation. Thus, A-PRF might influence bone and soft
tissue regeneration, especially through the presence of monocytes/macrophages and their growth factors. The
relevance and feasibility of this tissue-engineering concept have to be proven through in vivo studies.

Key Words: PRF, neutrophils, inflammation, tissue engineering, platelets, macrophages

INTRODUCTION
1
FORM – Frankfurt Orofacial Regenerative Medicine, Clinic of

A
Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Medical major objective of biomaterial re-
Center of the Goethe University Frankfurt, Frankfurt am Main,
Germany.
search and tissue engineering is to
2
RepairLab, Institute of Pathology, University Medical Center of promote a material-induced tissue
the Johannes Gutenberg University Mainz, Mainz, Germany.
3
Restorative Dentistry, School of Dental Medicine, State
reaction that leads to regeneration
University of New York – Buffalo, Buffalo, NY. and an effective wound-healing pro-
4
Private practice, Pain Therapy Center, Nice, France.
* Corresponding author, e-mail: shahram.ghanaati@kgu.de
cess in the defective area. Thus, a biomaterial
DOI: 10.1563/aaid-joi-D-14-00138 should serve as a temporary barrier to cover defects

Journal of Oral Implantology 679

Cell-Based Tissue Engineering by Means of Inflammatory Cells
and promote tissue regeneration while being tissue
compatible and, most importantly, clinically appli-
cable. In the field of tissue regeneration, vascular-
ization plays a crucial role as it ensures a continuous
supply of nutrients to and the removal of waste
products from the scaffold and the transplanted
region.
Concepts such as the use of a biomaterial
alone1,2 or preseeded with different primary mes-
enchymal3 or endothelial cells4,5 are usual prereq-
uisites for clinically applicable tissue engineering.
However, concepts involving the precultivation of
cells require time for cell isolation or cultivation as
well as the possibility to aseptically handle more
complex constructs in the operating room. These
become major challenges if there is demand for a
fast, robust, and ‘‘simple’’ approach by means of
cell-based tissue engineering. Obviously, time is one
of the most precious commodities in the clinic.
To ensure that methods for tissue engineering
are widely applicable in the clinical field, it is
necessary to modify them in a way that they are
readily available and relatively easy to use within
the daily clinical routine. Therefore, the steps
between the preparation and application have to
be minimized and optimized to make practical
implementation realistic. Thus, it is the overall goal
to develop concepts that are of natural origin, can
be produced ‘‘close’’ to the patients, and would
expedite the process of implantation while being
financially realistic for the patient and the health
system.
The requirements mentioned above led us to
look for new strategies in which a new class of
biomaterials is generated from autologous blood.6–9
Platelet-rich plasma (PRP) was the first generation
scaffold derived from human blood samples, and
this concept was widely studied and established
with common denominators such as the addition of
anticoagulants and bovine serum and double/-
centrifugation.10 Comparative studies showed that
PRP has a positive effect on the wound-healing
process and tissue regeneration.10 However, the
addition of anticoagulants and bovine serum limits
the clinical application of PRP and calls for
alternative, clinically feasible strategies.
With the aim of improving and streamlining
these preparation methods—especially towards
cell-seeded biomaterials generated from the pa-
tient’s own blood—a concept of platelet-rich fibrin
680 Vol. XL /No. Six /2014
(PRF) was developed.11–16 This fibrin scaffold, which
does not possess any cytotoxic potential,17 is
obtained from 9 mL of the patient’s own blood
after 1 centrifugation step. The factors previously
used in the preparation of blood-based scaffolds,
such as anticoagulants or bovine serum, were
excluded from this preparation procedure, which
minimized the risk of trans-contamination. The main
approach was to keep the methodology convenient
and applicable for clinical use.11–16 The three-
dimensional fibrin network is capable of mimicking
the extracellular matrix in terms of its structure,18,19
which creates the environment for cells to function
optimally.
Choukroun’s PRF is derived from human blood
and contains a variety of blood cells—including
platelets, B- and T-lymphocytes, monocytes, stem
cells, and neutrophilic granulocytes—as well as
growth factors.20,21 A major advantage of this
method is its simplicity of preparation. The centri-
fugation process activates the coagulation process
and as a result the clot is formed. This clot consists
of a 3-dimensional fibrin network in which the
platelets and other blood cells are entrapped. The
release of growth factors from the PRF clot
commences 5 to 10 minutes after clotting and
continues for at least 60 to 300 minutes.21,22 Several
studies with Choukroun’s PRF have shown the
tissue regenerative potential of this cell-loaded 3-
dimensional scaffold. Interestingly, this scaffold is
also a carrier for mesenchymal cells. Ling He et al
were able to show that different cell types, such as
rat osteoblasts, could differentiate and proliferate
when cultured on the leukocyte-rich PRF (L-PRF).
Differentiation and proliferation rates were investi-
gated in terms of transforming-growth factor b
(TGF-b), platelet-derived-growth factor AB (PDGF-
AB), and alkaline phosphatase (ALP) activity at 5
time points. The study showed a slow but constant
release of TGF-b1 and PDGF-AB as well as ALP
activity.23
Further experimental studies demonstrated that
even dermal pre-keratinocytes, human gingival
fibroblasts, pre-adipocytes, and maxillofacial osteo-
blasts underwent differentiation and proliferated
with Choukroun’s PRF. Dental pulp cells were also
able to grow and undergo further ‘‘differentiation’’
on the fibrin scaffold.24 In addition to the exper-
imental approaches discussed above, clinical appli-
cability of the material was tested. Mazor et al

FIGURE 1. Clots after centrifugation: (a) depicts a platelet-
rich fibrin (PRF) clot removed from the centrifugation tube
with forceps and placed on sterile gauze. (b) highlights
four PRF clots after careful removal of the red blood cell
(RBC) fraction count with scissors. The PRF clots are placed
in Choukroun’s PRF Box. The box is especially designed for
the processing of the PRF. It is composed of an outer lid, an
inner lid used for compressing the clots, a perforated plate
as seen in the picture, and a bottom used for conserving
the exudated fluid. As seen in the image on the right-hand
side, the two sockets are used to compress the clots, if
needed.
showed that biopsies of the augmented/implant
area taken 6 months after implantation revealed
new bone formation, thus highlighting its possible
osteo-inductive potential.25
Although the studies mentioned above under-
line the considerable potential of PRF in term of
tissue regeneration and clinical application, it was
still not clear how cells are distributed in this type
of scaffold depending on varying centrifugation
time and speed (ie, cumulative centrifugational
force). The aim of the study was to assess
Ghanaati et al
histologically and histomorphometrically the cell
distribution pattern of fibrin clots by applying the
standard PRF (S-PRF) protocol with 12-minute
centrifugation time and 2700 rpm as well as the
new advanced PRF (A-PRF) protocol with 14-
minute centrifugation time and 1500 rpm.
MATERIAL AND METHODS
Production of Choukroun’s PRF
The PRF scaffolds were prepared according to a
previously published protocol.11,26 Four healthy (ie,
with no history of anticoagulant usage) volunteers
in an age range between 18 to 60 years participated
in this study. For each individual, 4 tubes of
peripheral blood were collected and immediately
placed in a preprogrammed centrifuge (PC-O2,
PROCESS for PRF, Nice, France). Centrifugation was
performed according to the following two proto-
cols: (1) standard PRF, sterile glass coated plastic
tube (9 mL; 2700 rpm for 12 minutes) and (2)
advanced PRF, sterile plain glass-based vacuum
tubes (A-PRF10 tube) (10 mL; 1500 rpm for 14
minutes).
Histological preparation
Tissue Processing
After centrifugation, the clots were carefully re-
trieved from the tubes. The red blood cell (RBC)
fraction was removed in such a manner that the
bottom of the fibrin-rich clot was not damaged. This
technique is described in more detail in the
Choukroun protocol.11,26 An impression about the
shape of the clot is given in Figure 1. Fibrin-based
clots with the buffy coat (BC) and part of the RBC
were subsequently fixed with 4% paraformaldehyde
solution for 24 hours. After that, they were cut and
placed along the longitudinal axis into embedding
cassettes.
Histology and Histochemistry
For further microscopic analysis, the samples were
chemically processed in an alcohol series and xylene
as previously described.1,2 Subsequently, paraffin
embedding was performed, and 10 sections of 2 to
4 lm thickness, were cut with a rotary microtome
(Leica RM2255, Wetzlar, Germany) and affixed on
charged glass slides (SuperFrost Plus, Thermo
Scientific, Waltham, Mass). Before staining, samples
Journal of Oral Implantology 681
Cell-Based Tissue Engineering by Means of Inflammatory Cells

TABLE
Immunohistochemical markers and the specification of their use in the present study
Type
Antibody Targeted cell Clone (mono or poly) Epitope demasking Concentration
Anti-CD3 T-lymphocytes — Polyclonal rabbit Tris-EDTA, pH 8.0 1:200
anti-human
Anti-CD15, in accordance to reference27 Neutrophilic Carb-3 Monoclonal mouse Tris-EDTA, pH 8.0 RTU
granulocytes anti- human
Anti-CD20cy B-lymphocytes Clone L26 Monoclonal Mouse Tris-EDTA, pH 8.0 1:1000
anti-human
Anti-CD34 class II Stem cells Clone Monoclonal Mouse Tris-EDTA, pH 8.0 RTU
QBend-10 anti- human
Anti-CD61 platelet glycoprotein IIIa Platelets Y2/51 Monoclonal Mouse Tris-EDTA, pH 8.0 1:500
anti-human
Anti-CD68 Monocyte KP1 Monoclonal Mouse Tris-EDTA, pH 8.0 RTU
anti-human

underwent a deparaffinization and rehydratation
process by sequential immersion in xylene followed
by descending concentrations of ethanol. Several
histochemical and immunohistochemical staining
methods were then performed, as described as
follows:
Three samples were histologically stained with
standard protocols for hematoxylin and eosin (H&E)
and Masson-Goldner’s trichome technique, as these
special histochemical staining methods allowed
discrimination of the components of the clots, that
is, between cells and matrix proteins such as
fibrin.1,2 The other six sections were used to identify
various markers (Table) by means of immunohisto-
chemical staining in an autostainer (DAKO, Ham-
burg, Germany) at the Institute of Pathology,
Johann Wolfgang Goethe University Frankfurt,
Germany.
Immunohistochemistry
After establishing the optimal antibody concen-
tration for each of the above-mentioned markers
(DAKO), the slides were placed in a rack and
incubated in Tris-EDTA pH 8.0 at 968C for 20
minutes. Subsequently, the slides were rinsed with
running tap water to cool. Then, the slides were
washed with TBS and transferred to the autos-
tainer. Before starting the autostainer, the re-
quired antibodies and solutions were loaded into
the autostainer according to the manufacturer’s
instructions. The DAKO EnVision detection system
was used. After autostaining, the slides were
counterstained with hemalun for 30 seconds and
washed with running tap water. Finally, the
stained slides were cover-slipped with Aquatex
water-based, mounting medium (Merck, Ger-
many).
Histological evaluation
Histological examination was performed using a
light microscope (Nikon Eclipse 80i, Tokyo, Japan).
High-resolution photographs were taken with a
connected Nikon DS-Fi1/Digital camera and a Nikon
Digital sight unit DS-L2.
Histomorphometry
For histomorphometrical analysis, the six immuno-
histochemically stained slides of each clot were
scanned by means of a Nikon Eclipse 80i micro-
scope fitted with an automatic scanning table
(Prior Scientific, Rockland, Maine), a Nikon DS-Fi/1
digital camera and a computer with the Nikon NIS
– Elements AR software, version 4.0. This combi-
nation allowed the capture of single high-resolu-
tion images and subsequent assembly of single
images to one complete image (ie, a ‘‘total scan’’).
The following histomorphometrical analyses of
the comparative cell penetration within the clots
were done also using the measurement function of
the NIS – Elements software. Therefore, the total
length of every clot was measured at first (in
micrometer), and after that, the ‘‘penetration’’
lengths of the following cell types were also
determined (in micrometer): stem cells, T- and B-
lymphocytes, neutrophilic granulocytes, platelets,
and monocytes. Finally, the distribution of the

682 Vol. XL /No. Six /2014


FIGURE 2. Total scan of a fibrin clot along its longitudinal
axis (Masson-Goldner staining). RBC represents the red
blood cell fraction. The buffy coat (BC) is the transforma-
tion zone between the RBC fraction and fibrin clot, and FC
represents the fibrin clot. The three bars within the scan
and the arrows show close-ups of the respective areas. The
red arrows mark cells that are entrapped within the fibrin
network.
different cell types was put into context within the
respective clot length, thus allowing expression of
the ‘‘penetration’’ lengths in percent (ie, ‘‘relative
cell penetration’’) for the subsequent statistical
comparisons.
Statistical evaluation
Statistical analysis was carried out by using the
data of the two different experimental groups (ie,
with n ¼ 4 samples in each group). Penetration
depths were then compared statistically. The
obtained data yielded a mean 6 S.E.M. with the
aid of 1-way analysis of variance and a Bonferroni
multiple comparisons post-hoc test via the Graph-
Pad Prism version 6.0 (GraphPad Software, La
Jolla, Calif). Thereby, inter-individual as well as
intra-individual statistical significance were report-
ed as significant (/*, respectively) at P , .05, and
as highly significant (/**) at P , .01 and (/***)
at P , .001. Finally, the quantitative data were
presented graphically as the mean 6 standard
deviation (SD).
Ghanaati et al
RESULTS
Histochemical studies (H&E, Mason-Goldner, and
Giemsa)
S-PRF
In the longitudinal section of the S-PRF clot,
produced according to the standard centrifugation
protocol (2700 rpm, 12 minutes), a dense fibrin clot
was seen with minimal interfibrous space. With the
standard histochemical staining methods, cells were
observed throughout the clot, albeit decreasing
toward the more distal parts of the PRF clot (data
not shown).
A-PRF
PRF clots formed with the A-PRF centrifugation
protocol (1500 rpm, 14 minutes) showed a looser
structure with more interfibrous space, and more
cells could be counted in the fibrin-rich clot.
Furthermore, the cells were more evenly distributed
throughout the clot as compared to S-PRF, and
some cells could be found even in the clot’s more
distal parts. A representative image for cell distri-
bution within A-PRF is given in Figure 2.
Descriptive immunohistochemical evaluation
S-PRF
The slides stained for the respective markers were
evaluated in terms of specific cell type distribution
(Figures 3 and 4). In general, the highest number of
positively labeled cells was present in close
proximity to the RBC or in the BC. In particular,
cells with positive immunolabeling—including T-
lymphocytes (CD3-positive cells), B-lymphocytes
(CD20-positive cells), stem cells (CD34-positive
cells), and monocytes (CD68-positive cells)—were
found at the transition zone of the RBC fraction, BC,
and proximal parts of the clot. The platelets (CD61-
positive cells) were distributed throughout the clot.
However, decreased numbers were observed from
the proximal (near the BC) to the distal part of the S-
PRF clot. Additionally, neutrophilic granulocytes
(CD15-positive) showed a tendency to accumulate
mainly in the RBC-BC clot interface.
A-PRF
Analogous to the S-PRF, slides were stained
immunohistochemically for the markers of interest
(Figures 3 and 4). Most of the CD3-, CD20-, CD34-,
and CD68-positive cells stained in or near the BC (ie,
Journal of Oral Implantology 683
Cell-Based Tissue Engineering by Means of Inflammatory Cells
FIGURE 3. Immunohistochemical reaction of the cells
detected within the clots of the two experimental groups.
Images S1–S6 show close-ups of the transformation zone
between red blood cell (RBC), buffy coat (BC), and fibrin clot
(FC) with different immunohistochemical staining for CD 3-,
684 Vol. XL /No. Six /2014
FIGURE 4. Two total scans of the standard platelet-rich fibrin
(PRF): left, hematoxylin and eosin (H&E) staining, total scan,
100 3 magnification, and the advanced PRF; right, Masson-
Goldner staining, total scan, 100 3 magnification. Between
both total scans, each immunohistochemical marker is
portrayed by a colored bar (CD34–CD61). Within each of the
total scans, the distribution of each cell type is depicted by
separately colored bars. By looking at the total scans of both
the standard and advanced PRF, it becomes apparent that
CD61-positive cells (platelets) are distributed evenly
throughout the clot, although the amount of platelets
appears to decrease toward the peripheral parts of the clot
in the S-PRF group. Additionally, the modification of the
protocol resulted in an increased migration of CD15-positive
cells into the A-PRF clot.
the very proximal part of the fibrin clot); however,
the BC was more extensive in comparison to S-PRF.
Additionally, the neutrophilic granulocytes (ie,
CD15-positive cells) were distributed more widely
toward the distal (ie, away from the BC) part of the
fibrin-clot. Approximately two-thirds of the clot was
seeded with neutrophilic granulocytes/CD15-posi-
tive cells, with only the last third (distal part of the
fibrin clot) spared. As has been observed in the S-
CD15-, CD20-, CD34-, CD61-, and CD68-positive cells in the
respective picture for platelet-rich fibrin (PRF) clots pro-
duced with the standard PRF protocol. As can be seen in the
different images, the stained cells are mainly located within
the border between the RBC count and the BC. Depending
on the immunohistochemical marker, the cells are more or
less prominent in the PRF clot. The red arrows mark the cells
stained cells (positive) within the respective picture. The
same applies for the Figure A1 through A6. These images
are taken from PRF clots produced with the advanced PRF
protocol.

FIGURE 5. Comparative penetration of the different cell types
into the clot for both the standard platelet-rich fibrin (S-PRF)
and advanced platelet-rich fibrin (A-PRF). As can be seen in
this diagram, a highly significant difference (**P , .01) in
distribution was established for neutrophilic granulocytes
(CD15) between both groups.
PRF group, platelets (ie, CD61-positive) were found
throughout the entire clot. When compared to the
S-PRF group, the amount of CD61-positive cells did
not decrease to the same extent in the periphery.
Quantitative histomorphometric analysis of cell
penetration
The histomorphometrical analysis of the total scans
permitted an evaluation and comparison of the
respective cell distribution in the PRF clots. The total
length of each clot was measured and a mean of 6
SEM was calculated. The distribution/allocation of
each cell type was evaluated in the corresponding
total scan of the immunohistochemical staining.
The analyses revealed that platelets were the only
ones found in each area of the clot up to 87 6 13%
in the S-PRF group and up to 84 6 16% in the A-PRF
group (Figure 5). Furthermore, the results showed
that T-lymphocytes (S-PRF: 12 6 5%, A-PRF: 17 6
9%), B-lymphocytes (S-PRF: 14 6 7%, A-PRF: 12 6
9%), CD34-positive stem cells (S-PRF: 17 6 6%, A-
PRF: 21 6 11%), and monocytes (S-PRF: 19 6 9%, A-
PRF: 22 6 8%) were not found beyond a certain
point of maximally 30% of the total clot length, as
they are distributed in or near the BC generated by
the centrifugation process (Figure 5). Statistical
analysis revealed no statistically significant differ-
ences between the values of the two groups
concerning the allocation of these cell types (Figure
5). It is a remarkable observation that the changes in
parameters in PRF centrifugation protocols result in
an increased cell distribution of neutrophilic gran-
ulocytes up to 68 6 24% of the scaffold within the
A-PRF group, while this cell type was located up to
Ghanaati et al
25 6 12% of the clot length in the S-PRF group
(Figure 5). In this case, statistical analysis showed a
highly significant difference regarding the ‘‘distri-
bution’’ depth of this cell type between both study
groups (**P , .01) (Figure 5).
Additionally, no significant differences in the
distribution depths between the platelets, T-
lymphocytes, B-lymphocytes, stem cells, and
monocytes were found when comparing the two
centrifugation steps (Figure 5). Furthermore, the
analyses showed that the distribution depth of the
neutrophilic granulocytes in the A-PRF group was
highly increased compared to the before-men-
tioned cell types (**P , .01), while no interindi-
vidual differences were found in the S-PRF group
between these cell types (Figure 5). Finally, the
distribution depths of the platelets were signifi-
cantly higher compared to the values of all other
cell types in both study groups (***P , .001)
(Figure 5).
DISCUSSION
Complex tissue engineering concepts have to be
evaluated in terms of their clinical applicability.
Thus, the overall goal should be to establish a
method that could ideally be completed within a
short time span before or during intended regen-
erative surgical procedures. Over the last few years,
Choukroun’s PRF has proven to be a method that
comes close to the ideal concept of guided
(‘‘smart’’) tissue engineering. As previously shown,
this form of PRF contains growth factors such as
TGF-b1 and PDGF-AB23 and can contribute to tissue
regeneration in terms of osteoblast, prekeratino-
cyte, and gingival fibroblast differentiation.18
Even though the experiments mentioned above
show the promising capacities of this concept in
terms of contribution to successful tissue regener-
ation, up until now, there have been no studies
showing the extent to which the cells are distrib-
uted into the fibrin scaffold in relation to the two
essential parameters centrifugation speed and
centrifugation time.
In the present work, we have shown that T-
lymphocytes, B-lymphocytes, stem cells, and mono-
cytes are found in both groups within the first 25–
30% proximal part of the clot. There are no
statistically significant differences between both
groups in terms of distribution of those cells.
Journal of Oral Implantology 685
Cell-Based Tissue Engineering by Means of Inflammatory Cells
The tissue regeneration or repair process re-
quires harmonious reaction of various types of cells,
including immune response cells (neutrophils,
macrophages, lymphocytes), epithelial cells, fibro-
blasts, and stem cells, as well as other cells relevant
for the tissue in question. The PRF scaffold concept
seems to be an ideal source of components for the
healing process. An autologous blood-derived
scaffold can be a unique source of hematopoietic
stem cells (HSCs), which are of major importance in
regenerative medicine. In the past decade, many
studies highlighted the vast differentiation potential
of HSCs.28,29 A recent review by Ogawa et al
presented strong evidence for the pluripotent
capacity of HSCs and summarized work that has
been done in this field.30 Apart from their ability to
replenish the majority of cell types in the body,
stem cells also play a role as immune modulators31:
They can target B-lymphocytes and stimulate
antibody secretion, inhibit or even lead to apoptosis
of T-lymphocytes, and induce immune tolerance.
Other cells that can be observed in these
advanced fibrin clots are B- and T-lymphocytes.
Lymphocytes are responsible for specific and
nonspecific intervention in tissue response for
injury, although they are not prominent in the first
phase of tissue repair. A study by Boyce et al
revealed that CD8þ T-lymphocytes decreased
wound healing, whereas B-lymphocytes were asso-
ciated with an increased healing.32,33
In contrast to the cells mentioned above,
platelets are distributed more evenly throughout
the entire clot. It appears that a decrease in
centrifugation speed and an increase in centrifuga-
tion time results in higher platelet concentrations in
the distal part of clot, although this observation was
not definitively proven. Platelets, providing the
name for these fibrin-rich scaffolds (ie, platelet-rich
fibrin) are not only present in the coagulation
pathway or primary wound closure but also have a
vast regenerative potential by releasing a broad
spectrum of cytokines, chemokines, growth factors,
and other mediators. Platelets are able to release,
amongst others, molecules such as von Willebrand
factor, P-selectin, fibronectin, VEGF, platelet-derived
endothelial growth factor (PDEGF), vitronectin, and
fibrinogen. With these different growth factors,
adhesion molecules, and other mediators, platelets
have the ability to initiate and modulate host
immune responsiveness through influencing neu-
686 Vol. XL /No. Six /2014
trophils, monocytes, and endothelial cells, as well as
lymphocytes. Upon stimulation, platelets actively
participate in pathogen detection, capturing, and
sequestration. They can even induce the death of
infected cellular targets. Thanks to this wide range
of activities, platelets are visible at each step of
regeneration.34
Monocytes are also essential for tissue healing.
They migrate into the inflamed area after the influx
of neutrophils, where they then become macro-
phages.35 Macrophages are multifunctional cells
that represent distinct phenotypes. They have
substantial roles in foreign body response, osteo-
genesis, and angiogenesis as they respond to
inserted biomaterials.36,37 Through expression of
VEGF, PDGF, FGF, TGFa and –b, and other biolog-
ically active molecules (eg, BMP-2), macrophages
support cell proliferation and tissue restoration
following injury.36,38 They are seen throughout all
the processes of tissue repair from early inflamma-
tion through tissue-remodeling and scar forma-
tion.38
One of the most important findings of the
present study is that changing the centrifugation
protocol in terms of centrifugation time and speed
leads to a different distribution pattern for neutro-
philic granulocytes. Neutrophilic granulocytes are
most commonly considered to be early inflamma-
tory cells due to their phagocytotic capacity,
degranulation, and neutrophilic extracellular
traps.40,41 However, recent studies have shown that
neutrophilic granulocytes have tissue regeneration
properties as well. Neutrophils also facilitate traf-
ficking of monocytes into the wound to phagocy-
tose inflammatory remnants (necrotic and apoptot-
ic cells).40,42 Moreover, they also participate in the
process of wound debridement by secreting several
proteases, including matrix metalloproteinase 9
(MMP9), an extracellular matrix digesting enzyme.
Furthermore, neutrophilic granulocytes expressing
MMP9 play a part in the process of revascularization
of the tissue defect by being recruited, for example,
by VEGF-A.17,40 Neutrophilic granulocytes and
monocytes/macrophages are in mutual communi-
cation, and their interplay contributes to further
differentiation towards a pro- or anti-inflammatory
state of the macrophages. Additionally, recent
studies of Tan et al revealed a potential contribution
of neutrophils to inflammatory lymphogenesis by
VEGF-A modulation and VEGF-D secretion in a

murine model.43 These cells modulate the innate as
well as adaptive immune response in a direct and
indirect manner by crosstalk with B- and T-
lymphocytes.44 Thus, the distribution of neutrophil-
ic granulocytes within the A-PRF clot might be the
basis for a better functionality of the transplanted
(but also resident) monocytes/macrophages and
lymphocytes and their deployment to support
tissue regeneration.
Aside from the neutrophilic granulocyte’s role in
early inflammation and its potential regenerative
properties, it has to be questioned why granulo-
cytes are capable of migrating deeper into the
matrix of the clot. One approach to addressing this
question is the neutrophilic diameter. Thus, with an
average diameter of 8.5–10 lm neutrophilic gran-
ulocytes are remarkably smaller than monocytes
(diameter of 15–20 lm45) and therefore could be
more prone to penetrate deeper into the clot
during the centrifugation process. If this, however,
is true, it needs to be determined whether the
weight of neutrophils correlates with their diameter.
Basically, differential centrifugation is based on the
differences in the sedimentation rate of the
(biological) particles of difference size, shape, and
density, as well as conditions of centrifugation.
However, this present study failed to uncover why
the neutrophilic granulocytes, in particular, ‘‘react’’
toward modifications in the centrifugation protocol.
Therefore, further research has to be performed to
validate these findings and give a scientific expla-
nation for the different neutrophil behavior.
Among the ideas that are presently being
established in the field of tissue regeneration—
such as the use of co-cultures or monocultures, for
example with osteoblasts or fibroblasts3—the PRF
concept could be an additional component suit-
able for clinical applications. This concept of
generating a fibrin-based cell-seeded matrix solely
by drawing blood and centrifugation for 12–14
minutes is truly revolutionary in terms of clinical
practicability, as it can be handled and modified
easily in a short period of time and provides the
defect not only with a matrix permitting cell
migration into the defected area but also providing
the wound with crucial biological cues, potentially
accelerating the wound-healing process: These
include platelet-derived growth factor (PDGF),
transforming growth factor b (TGF-b), platelet
factor 4 (PF4), IL-1, vascular endothelial growth
Ghanaati et al
factor (VEGF), epidermal growth factor, endothelial
cell growth factor (ECGF), platelet-derived endo-
thelial growth factor (PDEGF), insulin-like growth
factor, osteocalcin, osteonectin, fibrinogen, vitro-
nectin, fibronectin, and thrombospondin.6 Further-
more, the fact that it is a matrix that is generated by
the patient’s own (ie, autologous) blood, there is
virtually no risk of a rejection reaction (foreign body
response).
The future of clinically applicable tissue engi-
neering concepts will most likely be spearheaded
by cell-based tissue constructs that can be gener-
ated in a short time span and in close proximity to
the patient. Nevertheless, it still has to be deter-
mined how these constructs, including Choukroun’s
variation of PRF, will interact in vitro and in vivo
after combination with cultured osteoblasts, fibro-
blasts, or a mixture with osteoconductive granules
in vivo. It still remains to be shown in well-designed
clinical studies that this fibrin-based matrix can
facilitate guided bone-tissue engineering with
simultaneous avoidance of obstacles such as
infection, rejection, and cumbersome isolation
methods. It is hoped that such clinical studies will
also reveal to what extent the neutrophilic granu-
locytes contribute to the regenerative properties of
the PRF.
CONCLUSION
The present data show that specific cell types are
distributed differentially depending on the (cumu-
lative) centrifugal force. This concept enables the
optimal scaffold or composites to be tailored for
specific clinical applications. These powerful com-
posites can contribute to wound healing and tissue
repair as well as tissue regeneration. Additionally, A-
PRF appears to be an ideal provider of autologous
cells (especially neutrophils and macrophages), thus
enabling mutual stimulation, thereby creating a
synergistic relationship in the interest of tissue
regeneration.
ABBREVIATIONS
ALP: alkaline phosphatase
ANOVA: analysis of variance
A-PRF: advanced platelet-rich fibrin
BC: buffy coat
H&E: hematoxylin and eosin
Journal of Oral Implantology 687
Cell-Based Tissue Engineering by Means of Inflammatory Cells
HSC: hematopoietic stem cells
L-PRF: leukocyte- and platelet-rich fibrin
PDEGF: platelet-derived endothelial growth factor
PDGF-AB: platelet-derived growth factor AB
PRF: platelet-rich fibrin
PRP: platelet-rich plasma
RBC: red blood cell
S-PRF: standard platelet-rich fibrin
TGF-b: transforming growth factor b
VEGF: vascular endothelial growth factor
ACKNOWLEDGMENT
The authors would like to thankfully acknowledge
the superb technical assistance of Ralf Liberz and
Yvonne Michel, Senckenberg Institute of Pathology,
Goethe University, Frankfurt am Main, Germany.
REFERENCES
1. Ghanaati S, Orth C, Unger RE, et al. Fine-tuning scaffolds for
tissue regeneration: effects of formic acid processing on tissue
reaction to silk fibroin. J Tissue Eng Regen Med. 2010; 4:464–472.
2. Ghanaati S, Schlee M, Webber MJ, et al. Evaluation of the
tissue reaction to a new bilayered collagen matrix in vivo and its
translation to the clinic. Biomed Mater. 2011;6:15010.
3. Ghanaati S, Unger RE, Webber MJ, JA et al. Scaffold
vascularization in vivo driven by primary human osteoblasts in
concert with host inflammatory cells. Biomaterials. 2011;32:8150–
8160.
4. Fuchs S, Ghanaati S, Orth C, et al. Contribution of
outgrowth endothelial cells from human peripheral blood on in
vivo vascularization of bone tissue engineered constructs based on
starch polycaprolactone scaffolds. Biomaterials. 2009;30:526–534.
5. Unger RE, Ghanaati S, Orth C, et al. The rapid anastomosis
between prevascularized networks on silk fibroin scaffolds gener-
ated in vitro with cocultures of human microvascular endothelial
and osteoblast cells and the host vasculature. Biomaterials. 2010;31:
6959–6967.
6. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a
review of biology and applications in plastic surgery. Plast Reconstr
Surg. 2006;118:147e–159e.
7. Bhanot S, Alex JC. Current applications of platelet gels in
facial plastic surgery. Facial Plast Surg. 2002;18:27–33.
8. Marques LF, Stessuk T, Camargo ICC, Sabeh Junior N,
Santos LD, Ribeiro-Paes JT. Platelet-rich plasma (PRP): methodo-
logical aspects and clinical applications. Platelets. In press.
9. Roubelakis MG, Trohatou O, Roubelakis A, et al. Platelet-
rich plasma (PRP) promotes fetal mesenchymal stem/stromal cell
migration and wound healing process. Stem Cell Rev. 2014;10:417–
428.
10. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T.
Classification of platelet concentrates: from pure platelet-rich
plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends
Biotechnol 2009;27:158–167.
11. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part I: technolog-
ical concepts and evolution. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod. 2006;101:e37–e44.
688 Vol. XL /No. Six /2014
12. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part II: platelet-
related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod. 2006;101:e45–e50.
13. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part III: leucocyte
activation: a new feature for platelet concentrates? Oral Surg Oral
Med Oral Pathol Oral Radiol Endod. 2006;101:e51–e55.
14. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part IV: clinical
effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod. 2006;101:e56–e60.
15. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part V: histologic
evaluations of PRF effects on bone allograft maturation in sinus lift.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:299–303.
16. Choukroun J, Adda F, Schoeffler C, Vervelle A. An
opportunity in paro-implantology: PRF. [in French]. Implantodontie.
2001;42:55–62.
17. Christoffersson G, Vågesjö E, Vandooren J, et al. VEGF-A
recruits a proangiogenic MMP-9-delivering neutrophil subset that
induces angiogenesis in transplanted hypoxic tissue. Blood. 2012;
120:4653–4662.
18. Dohan Ehrenfest DM, Diss A, Odin G, Doglioli P, Hippolyte
M, Charrier J. In vitro effects of Choukroun’s PRF (platelet-rich fibrin)
on human gingival fibroblasts, dermal prekeratinocytes, preadipo-
cytes, and maxillofacial osteoblasts in primary cultures. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2009;108:341–352.
19. Dohan Ehrenfest DM, Bielecki T, Jimbo R, et al. Do the fibrin
architecture and leukocyte content influence the growth factor
release of platelet concentrates? An evidence-based answer
comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte-
and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol. 2012;13:
1145–1152.
20. Kawazoe T, Kim HH. Tissue augmentation by white blood
cell-containing platelet-rich plasma. Cell Transplant. 2012;21:601–607.
21. Perut F, Filardo G, Mariani E, et al. Preparation method and
growth factor content of platelet concentrate influence the
osteogenic differentiation of bone marrow stromal cells. Cytother-
apy. 2013;15:830–839.
22. Su CY, Kuo YP, Nieh H, Tseng YH, Burnouf T. Quantitative
assessment of the kinetics of growth factors release from platelet
gel. Transfusion. 2008;48:2414–2420.
23. He L, Lin Y, Hu X, Zhang Y, Wu H. A comparative study of
platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect
of proliferation and differentiation of rat osteoblasts in vitro. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108:707–713.
24. Huang F, Yang S, Zhao J, Chang Y. Platelet-rich fibrin
increases proliferation and differentiation of human dental pulp
cells. J Endod. 2010;36:1628–1632.
25. Mazor Z, Horowitz RA, Del Corso M, Prasad HS, Rohrer MD,
Dohan Ehrenfest DM. Sinus floor augmentation with simultaneous
implant placement using Choukroun’s platelet-rich fibrin as the
sole grafting material: a radiologic and histologic study at 6
months. J Periodontol. 2009;80:2056–2064.
26. Dohan Ehrenfest DM. How to optimize the preparation of
leukocyte- and platelet-rich fibrin (L-PRF, Choukroun’s technique)
clots and membranes: introducing the PRF Box. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod. 2010;110:275–278; author reply 278–
280.
27. Nakayama F, Nishihara S, Iwasaki H, et al. CD15 expression
in mature granulocytes is determined by alpha 1,3-fucosyltransfer-
ase IX, but in promyelocytes and monocytes by alpha 1,3-
fucosyltransferase IV. J Biol. Chem. 2001;276:16100–16106.
28. Lim WF, Inoue-Yokoo T, Tan KS, Lai MI, Sugiyama D.

Hematopoietic cell differentiation from embryonic and induced
pluripotent stem cells. Stem Cell Res Ther. 2013;4:71.
29. Nicolaidou V, Wong MM, Redpath AN, et al. Monocytes
induce STAT3 activation in human mesenchymal stem cells to
promote osteoblast formation. PLoS ONE. 2012;7:e39871.
30. Ogawa M, LaRue AC, Mehrotra M. Hematopoietic stem cells
are pluripotent and not just ‘‘hematopoietic.’’ Blood Cells Mol Dis.
2013;51:3–8.
31. Wang L, Zhao Y, Shi S. Interplay between mesenchymal
stem cells and lymphocytes: implications for immunotherapy and
tissue regeneration. J Dent Res. 2012;91:1003–1010.
32. Boyce DE, Jones WD, Ruge F, Harding KG, Moore K. The
role of lymphocytes in human dermal wound healing. Br J
Dermatol. 2000;143:59–65.
33. Schäffer M, Barbul A. Lymphocyte function in wound
healing and following injury. Br J Surg. 1998;85:444–460.
34. Jenne CN, Urrutia R, Kubes P. Platelets: bridging hemosta-
sis, inflammation, and immunity. Int J Lab Hematol. 2013;35:254–
261.
35. Soltan M, Rohrer MD, Prasad HS. Monocytes: super cells for
bone regeneration. Implant Dent. 2012;21:13–20.
36. Ekström K, Omar O, Granéli C, Wang X, Vazirisani F,
Thomsen P. Monocyte exosomes stimulate the osteogenic gene
expression of mesenchymal stem cells. PLoS ONE. 2013;8:e75227.
Ghanaati et al
37. Maciel J, Oliveira MI, Colton E, et al. Adsorbed fibrinogen
enhances production of bone- and angiogenic-related factors by
monocytes/macrophages. Tissue Eng Part A. 2014;20:250–263.
38. Pirraco RP, Reis RL, Marques AP. Effect of monocytes/
macrophages on the early osteogenic differentiation of hBMSCs. J
Tissue Eng Regen Med. 2013;7:392–400.
39. Koh TJ, DiPietro LA. Inflammation and wound healing: the
role of the macrophage. Expert Rev Mol Med. 2011;13:e23.
40. Kolaczkowska E, Kubes P. Neutrophil recruitment and
function in health and inflammation. Nat Rev Immunol. 2013;13:
159–175.
41. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil
extracellular traps kill bacteria. Science. 2004;303:1532–1535.
42. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to
the site of inflammation: the leukocyte adhesion cascade updated.
Nat Rev Immunol. 2007;7:678–689.
43. Tan KW, Chong SZ, Wong FHS, et al. Neutrophils contribute
to inflammatory lymphangiogenesis by increasing VEGF-A bioavail-
ability and secreting VEGF-D. Blood. 2013;122:3666–3677.
44. Mócsai A. Diverse novel functions of neutrophils in
immunity, inflammation, and beyond. J Exp Med. 2013;210:1283–
1299.
45. Welsch U. Lehrbuch Histologie. 3rd ed. Munich: Elsevier,
Urban & Fischer; 2010.
Journal of Oral Implantology 689