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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.
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
and promote tissue regeneration while being tissue (PRF) was developed.11–16 This fibrin scaffold, which
compatible and, most importantly, clinically appli- does not possess any cytotoxic potential,17 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
alone1,2 or preseeded with different primary mes- approach was to keep the methodology convenient
enchymal3 or endothelial cells4,5 are usual prereq- and applicable for clinical use.11–16 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
become major challenges if there is demand for a and contains a variety of blood cells—including
fast, robust, and ‘‘simple’’ approach by means of platelets, B- and T-lymphocytes, monocytes, stem
cell-based tissue engineering. Obviously, time is one cells, and neutrophilic granulocytes—as well as
of the most precious commodities in the clinic. growth factors.20,21 A major advantage of this
To ensure that methods for tissue engineering method is its simplicity of preparation. The centri-
are widely applicable in the clinical field, it is fugation process activates the coagulation process
necessary to modify them in a way that they are and as a result the clot is formed. This clot consists
readily available and relatively easy to use within of a 3-dimensional fibrin network in which the
the daily clinical routine. Therefore, the steps platelets and other blood cells are entrapped. The
between the preparation and application have to release of growth factors from the PRF clot
be minimized and optimized to make practical commences 5 to 10 minutes after clotting and
implementation realistic. Thus, it is the overall goal continues for at least 60 to 300 minutes.21,22 Several
to develop concepts that are of natural origin, can studies with Choukroun’s PRF have shown the
be produced ‘‘close’’ to the patients, and would tissue regenerative potential of this cell-loaded 3-
expedite the process of implantation while being dimensional scaffold. Interestingly, this scaffold is
financially realistic for the patient and the health also a carrier for mesenchymal cells. Ling He et al
system. were able to show that different cell types, such as
The requirements mentioned above led us to rat osteoblasts, could differentiate and proliferate
look for new strategies in which a new class of when cultured on the leukocyte-rich PRF (L-PRF).
biomaterials is generated from autologous blood.6–9 Differentiation and proliferation rates were investi-
Platelet-rich plasma (PRP) was the first generation gated in terms of transforming-growth factor b
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 ‘‘differentiation’’
these preparation methods—especially towards on the fibrin scaffold.24 In addition to the exper-
cell-seeded biomaterials generated from the pa- imental approaches discussed above, clinical appli-
tient’s own blood—a concept of platelet-rich fibrin cability of the material was tested. Mazor et al
Histological preparation
Tissue Processing
FIGURE 1. Clots after centrifugation: (a) depicts a platelet-
rich fibrin (PRF) clot removed from the centrifugation tube
After centrifugation, the clots were carefully re-
with forceps and placed on sterile gauze. (b) highlights trieved from the tubes. The red blood cell (RBC)
four PRF clots after careful removal of the red blood cell fraction was removed in such a manner that the
(RBC) fraction count with scissors. The PRF clots are placed bottom of the fibrin-rich clot was not damaged. This
in Choukroun’s PRF Box. The box is especially designed for
technique is described in more detail in the
the processing of the PRF. It is composed of an outer lid, an
inner lid used for compressing the clots, a perforated plate Choukroun protocol.11,26 An impression about the
as seen in the picture, and a bottom used for conserving shape of the clot is given in Figure 1. Fibrin-based
the exudated fluid. As seen in the image on the right-hand clots with the buffy coat (BC) and part of the RBC
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 lm 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
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 washed with running tap water. Finally, the
process by sequential immersion in xylene followed stained slides were cover-slipped with Aquatex
by descending concentrations of ethanol. Several water-based, mounting medium (Merck, Ger-
histochemical and immunohistochemical staining many).
methods were then performed, as described as
follows: Histological evaluation
Three samples were histologically stained with Histological examination was performed using a
standard protocols for hematoxylin and eosin (H&E) light microscope (Nikon Eclipse 80i, Tokyo, Japan).
and Masson-Goldner’s trichome technique, as these High-resolution photographs were taken with a
special histochemical staining methods allowed connected Nikon DS-Fi1/Digital camera and a Nikon
discrimination of the components of the clots, that Digital sight unit DS-L2.
is, between cells and matrix proteins such as
fibrin.1,2 The other six sections were used to identify Histomorphometry
various markers (Table) by means of immunohisto-
For histomorphometrical analysis, the six immuno-
chemical staining in an autostainer (DAKO, Ham-
histochemically stained slides of each clot were
burg, Germany) at the Institute of Pathology,
scanned by means of a Nikon Eclipse 80i micro-
Johann Wolfgang Goethe University Frankfurt,
scope fitted with an automatic scanning table
Germany.
(Prior Scientific, Rockland, Maine), a Nikon DS-Fi/1
Immunohistochemistry digital camera and a computer with the Nikon NIS
– 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 scan’’).
incubated in Tris-EDTA pH 8.0 at 968C for 20 The following histomorphometrical analyses of
minutes. Subsequently, the slides were rinsed with the comparative cell penetration within the clots
running tap water to cool. Then, the slides were were done also using the measurement function of
washed with TBS and transferred to the autos- the NIS – Elements software. Therefore, the total
tainer. Before starting the autostainer, the re- length of every clot was measured at first (in
quired antibodies and solutions were loaded into micrometer), and after that, the ‘‘penetration’’
the autostainer according to the manufacturer’s lengths of the following cell types were also
instructions. The DAKO EnVision detection system determined (in micrometer): stem cells, T- and B-
was used. After autostaining, the slides were lymphocytes, neutrophilic granulocytes, platelets,
counterstained with hemalun for 30 seconds and and monocytes. Finally, the distribution of the
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.
FIGURE 2. Total scan of a fibrin clot along its longitudinal
Furthermore, the cells were more evenly distributed
axis (Masson-Goldner staining). RBC represents the red
blood cell fraction. The buffy coat (BC) is the transforma- throughout the clot as compared to S-PRF, and
tion zone between the RBC fraction and fibrin clot, and FC some cells could be found even in the clot’s more
represents the fibrin clot. The three bars within the scan distal parts. A representative image for cell distri-
and the arrows show close-ups of the respective areas. The bution within A-PRF is given in Figure 2.
red arrows mark cells that are entrapped within the fibrin
network. Descriptive immunohistochemical evaluation
different cell types was put into context within the S-PRF
respective clot length, thus allowing expression of The slides stained for the respective markers were
the ‘‘penetration’’ lengths in percent (ie, ‘‘relative evaluated in terms of specific cell type distribution
cell penetration’’) for the subsequent statistical (Figures 3 and 4). In general, the highest number of
comparisons. positively labeled cells was present in close
proximity to the RBC or in the BC. In particular,
Statistical evaluation cells with positive immunolabeling—including T-
lymphocytes (CD3-positive cells), B-lymphocytes
Statistical analysis was carried out by using the (CD20-positive cells), stem cells (CD34-positive
data of the two different experimental groups (ie, cells), and monocytes (CD68-positive cells)—were
with n ¼ 4 samples in each group). Penetration found at the transition zone of the RBC fraction, BC,
depths were then compared statistically. The and proximal parts of the clot. The platelets (CD61-
obtained data yielded a mean 6 S.E.M. with the positive cells) were distributed throughout the clot.
aid of 1-way analysis of variance and a Bonferroni However, decreased numbers were observed from
multiple comparisons post-hoc test via the Graph- the proximal (near the BC) to the distal part of the S-
Pad Prism version 6.0 (GraphPad Software, La PRF clot. Additionally, neutrophilic granulocytes
(CD15-positive) showed a tendency to accumulate
Jolla, Calif). Thereby, inter-individual as well as
mainly in the RBC-BC clot 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,
The tissue regeneration or repair process re- trophils, monocytes, and endothelial cells, as well as
quires harmonious reaction of various types of cells, lymphocytes. Upon stimulation, platelets actively
including immune response cells (neutrophils, participate in pathogen detection, capturing, and
macrophages, lymphocytes), epithelial cells, fibro- sequestration. They can even induce the death of
blasts, and stem cells, as well as other cells relevant infected cellular targets. Thanks to this wide range
for the tissue in question. The PRF scaffold concept of activities, platelets are visible at each step of
seems to be an ideal source of components for the regeneration.34
healing process. An autologous blood-derived Monocytes are also essential for tissue healing.
scaffold can be a unique source of hematopoietic They migrate into the inflamed area after the influx
stem cells (HSCs), which are of major importance in of neutrophils, where they then become macro-
regenerative medicine. In the past decade, many phages.35 Macrophages are multifunctional cells
studies highlighted the vast differentiation potential that represent distinct phenotypes. They have
of HSCs.28,29 A recent review by Ogawa et al substantial roles in foreign body response, osteo-
presented strong evidence for the pluripotent genesis, and angiogenesis as they respond to
capacity of HSCs and summarized work that has inserted biomaterials.36,37 Through expression of
been done in this field.30 Apart from their ability to VEGF, PDGF, FGF, TGFa and –b, and other biolog-
replenish the majority of cell types in the body, ically active molecules (eg, BMP-2), macrophages
stem cells also play a role as immune modulators31: support cell proliferation and tissue restoration
They can target B-lymphocytes and stimulate following injury.36,38 They are seen throughout all
antibody secretion, inhibit or even lead to apoptosis the processes of tissue repair from early inflamma-
of T-lymphocytes, and induce immune tolerance. tion through tissue-remodeling and scar forma-
Other cells that can be observed in these tion.38
advanced fibrin clots are B- and T-lymphocytes. One of the most important findings of the
Lymphocytes are responsible for specific and present study is that changing the centrifugation
nonspecific intervention in tissue response for protocol in terms of centrifugation time and speed
injury, although they are not prominent in the first leads to a different distribution pattern for neutro-
phase of tissue repair. A study by Boyce et al philic granulocytes. Neutrophilic granulocytes are
revealed that CD8þ T-lymphocytes decreased most commonly considered to be early inflamma-
wound healing, whereas B-lymphocytes were asso- tory cells due to their phagocytotic capacity,
ciated with an increased healing.32,33 degranulation, and neutrophilic extracellular
In contrast to the cells mentioned above, traps.40,41 However, recent studies have shown that
platelets are distributed more evenly throughout neutrophilic granulocytes have tissue regeneration
the entire clot. It appears that a decrease in properties as well. Neutrophils also facilitate traf-
centrifugation speed and an increase in centrifuga- ficking of monocytes into the wound to phagocy-
tion time results in higher platelet concentrations in tose inflammatory remnants (necrotic and apoptot-
the distal part of clot, although this observation was ic cells).40,42 Moreover, they also participate in the
not definitively proven. Platelets, providing the process of wound debridement by secreting several
name for these fibrin-rich scaffolds (ie, platelet-rich proteases, including matrix metalloproteinase 9
fibrin) are not only present in the coagulation (MMP9), an extracellular matrix digesting enzyme.
pathway or primary wound closure but also have a Furthermore, neutrophilic granulocytes expressing
vast regenerative potential by releasing a broad MMP9 play a part in the process of revascularization
spectrum of cytokines, chemokines, growth factors, of the tissue defect by being recruited, for example,
and other mediators. Platelets are able to release, by VEGF-A.17,40 Neutrophilic granulocytes and
amongst others, molecules such as von Willebrand monocytes/macrophages are in mutual communi-
factor, P-selectin, fibronectin, VEGF, platelet-derived cation, and their interplay contributes to further
endothelial growth factor (PDEGF), vitronectin, and differentiation towards a pro- or anti-inflammatory
fibrinogen. With these different growth factors, state of the macrophages. Additionally, recent
adhesion molecules, and other mediators, platelets studies of Tan et al revealed a potential contribution
have the ability to initiate and modulate host of neutrophils to inflammatory lymphogenesis by
immune responsiveness through influencing neu- VEGF-A modulation and VEGF-D secretion in a
murine model.43 These cells modulate the innate as factor (VEGF), epidermal growth factor, endothelial
well as adaptive immune response in a direct and cell growth factor (ECGF), platelet-derived endo-
indirect manner by crosstalk with B- and T- thelial growth factor (PDEGF), insulin-like growth
lymphocytes.44 Thus, the distribution of neutrophil- factor, osteocalcin, osteonectin, fibrinogen, vitro-
ic granulocytes within the A-PRF clot might be the nectin, fibronectin, and thrombospondin.6 Further-
basis for a better functionality of the transplanted more, the fact that it is a matrix that is generated by
(but also resident) monocytes/macrophages and the patient’s own (ie, autologous) blood, there is
lymphocytes and their deployment to support virtually no risk of a rejection reaction (foreign body
tissue regeneration. response).
Aside from the neutrophilic granulocyte’s role in The future of clinically applicable tissue engi-
early inflammation and its potential regenerative neering concepts will most likely be spearheaded
properties, it has to be questioned why granulo- by cell-based tissue constructs that can be gener-
cytes are capable of migrating deeper into the ated in a short time span and in close proximity to
matrix of the clot. One approach to addressing this the patient. Nevertheless, it still has to be deter-
question is the neutrophilic diameter. Thus, with an mined how these constructs, including Choukroun’s
average diameter of 8.5–10 lm neutrophilic gran- variation of PRF, will interact in vitro and in vivo
ulocytes are remarkably smaller than monocytes after combination with cultured osteoblasts, fibro-
(diameter of 15–20 lm45) and therefore could be blasts, or a mixture with osteoconductive granules
more prone to penetrate deeper into the clot in vivo. It still remains to be shown in well-designed
during the centrifugation process. If this, however, clinical studies that this fibrin-based matrix can
is true, it needs to be determined whether the facilitate guided bone-tissue engineering with
weight of neutrophils correlates with their diameter. simultaneous avoidance of obstacles such as
Basically, differential centrifugation is based on the infection, rejection, and cumbersome isolation
differences in the sedimentation rate of the methods. It is hoped that such clinical studies will
(biological) particles of difference size, shape, and also reveal to what extent the neutrophilic granu-
density, as well as conditions of centrifugation. locytes contribute to the regenerative properties of
However, this present study failed to uncover why the PRF.
the neutrophilic granulocytes, in particular, ‘‘react’’
toward modifications in the centrifugation protocol.
CONCLUSION
Therefore, further research has to be performed to
validate these findings and give a scientific expla- The present data show that specific cell types are
nation for the different neutrophil behavior. distributed differentially depending on the (cumu-
Among the ideas that are presently being lative) centrifugal force. This concept enables the
established in the field of tissue regeneration— optimal scaffold or composites to be tailored for
such as the use of co-cultures or monocultures, for specific clinical applications. These powerful com-
example with osteoblasts or fibroblasts3—the PRF posites can contribute to wound healing and tissue
concept could be an additional component suit- repair as well as tissue regeneration. Additionally, A-
able for clinical applications. This concept of PRF appears to be an ideal provider of autologous
generating a fibrin-based cell-seeded matrix solely cells (especially neutrophils and macrophages), thus
by drawing blood and centrifugation for 12–14 enabling mutual stimulation, thereby creating a
minutes is truly revolutionary in terms of clinical synergistic relationship in the interest of tissue
practicability, as it can be handled and modified regeneration.
easily in a short period of time and provides the
defect not only with a matrix permitting cell
ABBREVIATIONS
migration into the defected area but also providing
the wound with crucial biological cues, potentially ALP: alkaline phosphatase
accelerating the wound-healing process: These ANOVA: analysis of variance
include platelet-derived growth factor (PDGF), A-PRF: advanced platelet-rich fibrin
transforming growth factor b (TGF-b), platelet BC: buffy coat
factor 4 (PF4), IL-1, vascular endothelial growth H&E: hematoxylin and eosin
HSC: hematopoietic stem cells 12. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part II: platelet-
L-PRF: leukocyte- and platelet-rich fibrin related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol
PDEGF: platelet-derived endothelial growth factor Endod. 2006;101:e45–e50.
PDGF-AB: platelet-derived growth factor AB 13. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
(PRF): a second-generation platelet concentrate. Part III: leucocyte
PRF: platelet-rich fibrin
activation: a new feature for platelet concentrates? Oral Surg Oral
PRP: platelet-rich plasma Med Oral Pathol Oral Radiol Endod. 2006;101:e51–e55.
RBC: red blood cell 14. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin
S-PRF: standard platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical
effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol
TGF-b: transforming growth factor b Endod. 2006;101:e56–e60.
VEGF: vascular endothelial growth factor 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.
ACKNOWLEDGMENT 16. Choukroun J, Adda F, Schoeffler C, Vervelle A. An
The authors would like to thankfully acknowledge opportunity in paro-implantology: PRF. [in French]. Implantodontie.
2001;42:55–62.
the superb technical assistance of Ralf Liberz and 17. Christoffersson G, Vågesjö E, Vandooren J, et al. VEGF-A
Yvonne Michel, Senckenberg Institute of Pathology, recruits a proangiogenic MMP-9-delivering neutrophil subset that
Goethe University, Frankfurt am Main, Germany. 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)
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