CLINICAL

AdvancedPlatelet-RichFibrin: A NewConceptfor Cell-

BasedTissueEngineeringbyMeansofInflammatory
Cells 1
1,2

Shahram Ghanaati, MD, DMD *

Patrick Booms, PhD 1 3
Anna Orlowska, BSc, DVM
Alica Kubesch 1,2 1
Jonas Lorenz, 1
DDS 1,4
Jim Rutkowski, DMD, PhD
1
Constantin Landes, MD, DMD,
PhD Robert Sader, MD, DDS, PhD
CJ Kirkpatrick, MD, PhD, 2
DSc
Joseph Choukroun, MD

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

* Corresponding author, e-mail: [email protected]
DOI: 10.1563/aaid-joi-D-14-00138

A
1

3
FORM – Frankfurt Orofacial Regenerative Medicine, Clinic of
Oral,
4 Cranio-Maxillofacial and Facial Plastic Surgery, Medical
Center of the Goethe University Frankfurt, Frankfurt am Main,
Germany.
RepairLab, Institute of Pathology, University Medical Center of
the Johannes Gutenberg University Mainz, Mainz, Germany.
Restorative Dentistry, School of Dental Medicine, State
University of New York –Buffalo, Buffalo, NY.
Private practice, Pain Therapy Center, Nice, France.
 INTRODUCTION
major objective of
biomaterial re- search and
tissue engineering is to
promote a material-induced
tissue reaction that leads to
regeneration and an effective
wound-healing pro-
cess in the defective area. Thus, a
biomaterial should serve as a temporary
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Cell-Based Tissue Engineering by Means of Inflammatory Cells

11–16
and promote tissue regeneration while being tissue (PRF) was developed. This fibrin scaffold, which
17
compatible and, most importantly, clinically appli- does not possess any cytotoxic potential, is
cable. In the field of tissue regeneration, vascular- obtained from 9 mL of the patient’s own blood
ization plays a crucial role as it ensures a continuous after 1 centrifugation step. The factors previously
supply of nutrients to and the removal of waste used in the preparation of blood-based scaffolds,
products from the scaffold and the transplanted such as anticoagulants or bovine serum, were
region. excluded from this preparation procedure, which
Concepts such as the use of a biomaterial minimized the risk of trans-contamination. The main
1,2
alone or preseeded with different primary mes- approach was to keep the methodology convenient
3 4,5 11–16
enchymal or endothelial cells are usual prereq- and applicable for clinical use. The three-
uisites for clinically applicable tissue engineering. dimensional fibrin network is capable of mimicking
However, concepts involving the precultivation of the extracellular matrix in terms of its structure, 18,19
cells require time for cell isolation or cultivation as which creates the environment for cells to function
well as the possibility to aseptically handle more optimally.
complex constructs in the operating room. These Choukroun’s PRF is derived from human blood D
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become major challenges if there is demand for a and contains a variety of blood cells—including nl
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fast, robust, and ‘‘simple’’approach by means of platelets, B- and T-lymphocytes, monocytes, stem a
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cell-based tissue engineering. Obviously, time is one cells, and neutrophilic granulocytes—as well as df
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of the most precious commodities in the clinic. growth factors.20,21 A major advantage of this m
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To ensure that methods for tissue engineering method is its simplicity of preparation. The centri- tp
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are widely applicable in the clinical field, it is fugation process activates the coagulation process w
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necessary to modify them in a way that they are and as a result the clot is formed. This clot consists .j
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readily available and relatively easy to use within of a 3-dimensional fibrin network in which the o
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in
the daily clinical routine. Therefore, the steps platelets and other blood cells are entrapped. The e.
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between the preparation and application have to release of growth factors from the PRF clot g/
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be minimized and optimized to make practical commences 5 to 10 minutes after clotting and /p
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implementation realistic. Thus, it is the overall goal continues for at least 60 to 300 minutes.21,22 Several /1
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to develop concepts that are of natural origin, can studies with Choukroun’s PRF have shown the 1
5
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be produced ‘‘close’’to the patients, and tissue regenerative potential of this cell-loaded 3- 3/
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would expedite the process of implantation while dimensional scaffold. Interestingly, this scaffold is ai
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being financially realistic for the patient and the also a carrier for mesenchymal cells. Ling He et al i-
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health system. were able to show that different cell types, such as -
1
The requirements mentioned above led us to rat osteoblasts, could differentiate and proliferate 4-
0
0
look for new strategies in which a new class of when cultured on the leukocyte-rich PRF (L-PRF). 1
3
biomaterials is generated from autologous blood. 6–9 Differentiation and proliferation rates were investi- 8
b
Platelet-rich plasma (PRP) was the first generation gated in terms of transforming-growth factor b y
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scaffold derived from human blood samples, and (TGF-b), platelet-derived-growth factor AB (PDGF-
this concept was widely studied and established AB), and alkaline phosphatase (ALP) activity at 5
with common denominators such as the addition of time points. The study showed a slow but constant
anticoagulants and bovine serum and double/- release of TGF-b1 and PDGF-AB as well as ALP
centrifugation. 10 Comparative studies showed that activity.23
PRP has a positive effect on the wound-healing Further experimental studies demonstrated that
process and tissue regeneration.10 However, the even dermal pre-keratinocytes, human gingival
addition of anticoagulants and bovine serum limits fibroblasts, pre-adipocytes, and maxillofacial osteo-
the clinical application of PRP and calls for blasts underwent differentiation and proliferated
alternative, clinically feasible strategies. with Choukroun’s PRF. Dental pulp cells were also
With the aim of improving and streamlining able to grow and undergo further
these preparation methods—especially towards ‘‘differentiation’’ on24 the fibrin scaffold. In
cell-seeded biomaterials generated from the pa- addition to the exper- imental approaches
tient’s own blood—a concept of platelet-rich fibrin discussed above, clinical appli- cability of the
material was tested. Mazor et al
680 Vol. XL /No. Six /2014
 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 D
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in this study. For each individual, 4 tubes of w
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peripheral blood were collected and immediately o
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placed in a preprogrammed centrifuge (PC-O2, e
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PROCESS for PRF, Nice, France). Centrifugation was ro
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performed according to the following two proto- ht
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cols: (1) standard PRF, sterile glass coated plastic w
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tube (9 mL; 2700 rpm for 12 minutes) and (2) w
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advanced PRF, sterile plain glass-based vacuum o
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tubes (A-PRF10 tube) (10 mL; 1500 rpm for 14 in
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minutes). or
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Histological preparation /p
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Tissue Processing 1
5
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FIGURE 1. Clots after centrifugation: (a) depicts a platelet-
After centrifugation, the clots were carefully re- 3/
a
rich fibrin (PRF) clot removed from the centrifugation tube ai
trieved from the tubes. The red blood cell (RBC)
with forceps and placed on sterile gauze. (b) highlights d-
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fraction was removed in such a manner that the
four PRF clots after careful removal of the red blood cell i-
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bottom of the fibrin-rich clot was not damaged. This
(RBC) fraction count with scissors. The PRF clots are placed -
1
in Choukroun’s PRF Box. The box is especially designed for
technique is described in more detail in the 4-
0
the processing of the PRF. It is composed of an outer lid, an 0
Choukroun protocol.11,26 An impression about the
inner lid used for compressing the clots, a perforated plate
1
3
shape of the clot is given in Figure 1. Fibrin-based
as seen in the picture, and a bottom used for conserving
8
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clots with the buffy coat (BC) and part of the RBC
the exudated fluid. As seen in the image on the right-hand g
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side, the two sockets are used to compress the clots, if
were subsequently fixed with 4% paraformaldehyde
needed.
solution for 24 hours. After that, they were cut and
placed along the longitudinal axis into embedding
showed that biopsies of the augmented/implant cassettes.
area taken 6 months after implantation revealed
new bone formation, thus highlighting its possible Histology and Histochemistry
osteo-inductive potential. 25 For further microscopic analysis, the samples were
Although the studies mentioned above under- chemically processed in an alcohol series and xylene
line the considerable potential of PRF in term of as previously described.1,2 Subsequently, paraffin
tissue regeneration and clinical application, it was embedding was performed, and 10 sections of 2 to
still not clear how cells are distributed in this type 4 l m thickness, were cut with a rotary microtome
of scaffold depending on varying centrifugation (Leica RM2255, Wetzlar, Germany) and affixed on
time and speed (ie, cumulative centrifugational charged glass slides (SuperFrost Plus, Thermo
force). The aim of the study was to assess 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
anti-human Tris-EDTA, pH 8.0 1:200
Anti-CD15, in accordance to 27
Neutrophilic Carb-3 Monoclonal mouse Tris-EDTA, pH 8.0 RTU
reference
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 D
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underwent a deparaffinization and rehydratation washed with running tap water. Finally, the e
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process by sequential immersion in xylene followed stained slides were cover-slipped with Aquatex m
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by descending concentrations of ethanol. Several water-based, mounting medium (Merck, Ger- tp
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histochemical and immunohistochemical staining many). w
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methods were then performed, as described as .j

follows: Histological evaluation oi

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Three samples were histologically stained with Histological examination was performed using a
in
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standard protocols for hematoxylin and eosin (H&E) light microscope (Nikon Eclipse 80i, Tokyo, Japan). g/
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and Masson-Goldner’s trichome technique, as these High-resolution photographs were taken with a
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special histochemical staining methods allowed connected Nikon DS-Fi1/Digital camera and a Nikon /1
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discrimination of the components of the clots, that Digital sight unit DS-L2. 1
5
is, between
1,2
cells and matrix proteins such as 6
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fibrin. The other six sections were used to identify Histomorphometry ai
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various markers (Table) by means of immunohisto- jo
For histomorphometrical analysis, the six immuno- i-
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chemical staining in an autostainer (DAKO, Ham- -
histochemically stained slides of each clot were 1
burg, Germany) at the Institute of Pathology, 4-
scanned by means of a Nikon Eclipse 80i micro- 0
Johann Wolfgang Goethe University Frankfurt, 0
1
scope fitted with an automatic scanning table 3
Germany. 8
(Prior Scientific, Rockland, Maine), a Nikon DS-Fi/1 b
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Immunohistochemistry digital camera and a computer with the Nikon NIS g

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– Elements AR software, version 4.0. This combi-
After establishing the optimal antibody concen- nation allowed the capture of single high-resolu-
tration for each of the above-mentioned markers tion images and subsequent assembly of single
(DAKO), the slides were placed in a rack and images to one complete image (ie, a ‘‘total
incubated in Tris-EDTA pH 8.0 at 968C for 20 scan’’).
minutes. Subsequently, the slides were rinsed with The following histomorphometrical analyses of
running tap water to cool. Then, the slides were the comparative cell penetration within the clots
washed with TBS and transferred to the autos- were done also using the measurement function of
tainer. Before starting the autostainer, the re- the NIS – Elements software. Therefore, the total
quired antibodies and solutions were loaded into length of every clot was measured at first (in
the autostainer according to the manufacturer’s micrometer), and after that, the ‘‘penetration
instructions. The DAKO EnVision detection system ’’ lengths of the following cell types were
was used. After autostaining, the slides were also determined (in micrometer): stem cells, T-
counterstained with hemalun for 30 seconds and and B- lymphocytes, neutrophilic granulocytes,
platelets, and monocytes. Finally, the
682 Vol. XL /No. Six /2014 distribution of the
 Ghanaati et al

R ESULTS
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 D
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PRF clots formed with the A-PRF centrifugation nl
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protocol (1500 rpm, 14 minutes) showed a looser a
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structure with more interfibrous space, and more e
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cells could be counted in the fibrin-rich clot. m
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FIGURE 2. Total scan of a fibrin clot along its longitudinal Furthermore, the cells were more evenly distributed tp
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axis (Masson-Goldner staining). RBC represents the red throughout the clot as compared to S-PRF, and w
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blood cell fraction. The buffy coat (BC) is the transforma- w
tion zone between the RBC fraction and fibrin clot, and FC some cells could be found even in the clot’s more .j
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represents the fibrin clot. The three bars within the scan distal parts. A representative image for cell distri- o
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and the arrows show close-ups of the respective areas. The bution within A-PRF is given in Figure 2. in
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red arrows mark cells that are entrapped within the fibrin or
g/
network. Descriptive immunohistochemical evaluation d
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S-PRF /1
different cell types was put into context within the 0.
1
respective clot length, thus allowing expression of The slides stained for the respective markers were 5
6
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the ‘‘penetration’’lengths in percent (ie, ‘‘relative evaluated in terms of specific cell type distribution a
ai
cell penetration’’) for the subsequent statistical (Figures 3 and 4). In general, the highest number of d-
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comparisons. positively labeled cells was present in close i-

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proximity to the RBC or in the BC. In particular, cells 1
4-
Statistical evaluation with positive immunolabeling—including T-
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lymphocytes (CD3-positive cells), B-lymphocytes 1
3
8
Statistical analysis was carried out by using the (CD20-positive cells), stem cells (CD34-positive cells), b
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data of the two different experimental groups (ie, and monocytes (CD68-positive cells)—were found at g
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with n ¼ 4 samples in each group). Penetration the transition zone of the RBC fraction, BC, and proximal
depths were then compared statistically. The parts of the clot. The platelets (CD61- positive cells) were
obtained data yielded a mean 6 S.E.M. with the distributed throughout the clot. However, decreased
aid of 1-way analysis of variance and a Bonferroni numbers were observed from the proximal (near the BC)
to the distal part of the S- PRF clot. Additionally,
multiple comparisons post-hoc test via the Graph-
neutrophilic granulocytes (CD15-positive) showed a
Pad Prism version 6.0 (GraphPad Software, La
tendency to accumulate mainly in the RBC-BC clot
Jolla, Calif). Thereby, inter-individual as well as interface.
intra-individual statistical significance were report-
ed as significant ( /*, respectively) at P , .05, and A-PRF
as highly significant ( /**) at P , .01 and ( /***) Analogous to the S-PRF, slides were stained
at P , .001. Finally, the quantitative data were immunohistochemically for the markers of interest
presented graphically as the mean 6 standard (Figures 3 and 4). Most of the CD3-, CD20-, CD34-,
deviation (SD). and CD68-positive cells stained in or near the BC (ie,

Journal of Oral Implantology 683