Molecular Biology Reports (2021) 48:4573–4580

https://doi.org/10.1007/s11033-021-06487-7

ORIGINAL ARTICLE

Platelet activation and antimicrobial activity of L‑PRF: a preliminary

study
António Melo‑Ferraz1   · Cristina Coelho1   · Paulo Miller1   · Maria Begoña Criado1   · Maria Céu Monteiro1 

Received: 28 February 2021 / Accepted: 8 June 2021 / Published online: 19 June 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Leukocyte and platelet rich fibrin (L-PRF) is one of the platelet concentrates used to support regeneration and healing
process. Many studies showed possible immunological and antibacterial properties of L-PRF. We perform an in vitro study
to analyze the effect of L-PRF on platelet activation, platelet-leukocytes interactions and antimicrobial activity, important
components in the healing process. Molecular biomarkers related with platelet activation and platelet-leukocyte interactions
were analyzed by means of flow cytometry when L-PRF exudate was added to whole blood platelets. L-PRF membrane was
used to evaluate antimicrobial activity using Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853)
and Candida albicans (ATCC 90028). Our experimental design allows to evaluate platelet activation and analyze molecular
biomarkers of other immune cells and platelet-leukocyte interactions. From the results obtained we can conclude that L-PRF
can be a valuable tool in healing process, efficient in activating platelets of whole blood and inhibiting microbial growth. In
our opinion, the use of L-PRF exudate, in addition to L-PRF membrane, presents some advantages that have to be considered
in clinical trials. Additional research on the characterization and quantification of cells and its products present in the L-PRF
exudate, as well as on the temporal factor released. Also, further studies using strains isolated from clinical cases are needed.

Keywords  Autologous platelet concentrates · L-PRF · Whole blood platelets · Antimicrobial activity · Flow cytometry ·
Regenerative dentistry

Introduction studies have highlighted the important role of platelets in

Healing is a complex process whose underlying mechanisms
are not yet fully understood. However, it is established that
platelets play an important role in it by releasing different
growth factors [1, 2]. These factors are responsible for dif-
ferent effects such as an increase in collagen production, cell
mitosis, angiogenesis, recruitment and migration of cells to
the lesion site and induction of cell differentiation, among
others [3].
Several in vitro studies have shown that, after being stim-
ulated, platelets can also secrete proteins with antimicrobial
activity [4, 5], produce reactive oxygen species and par-
ticipate in antibody-dependent cell cytotoxicity [4]. Recent
* Maria Céu Monteiro
mceu.monteiro@ipsn.cespu.pt
1
IINFACTS – Institute of Research and Advanced Training
in Health Sciences and Technology, CESPU-Cooperativa de
Ensino Superior Politécnico e Universitário, Rua Central de
Gandra 1317, 4585 116 Gandra, PRD, Portugal
the recognition and neutralization of pathogens, as well as
their indirect contribution to the recruitment and modulation
of leukocyte behavior, increasing their ability to phagocyte
and eliminate microorganisms by activating different types
of intracellular signaling pathways [6].
One of the great current challenges in the clinic is to
make the healing process faster and to regulate inflammation
[7]. In this sense, the potential of platelet concentrates (PC)
in the regeneration process has been intensively studied in
recent years. Several studies have shown that PCs had anti-
inflammatory properties that resulted in a marked reduction
in postoperative pain and swelling. Applications of PCs in
tissue engineering in vivo include: (1) platelet-rich plasma
(PRP) and (2) platelet-rich fibrin (PRF). Regarding PRP,
its use appears to increase osteoprogenitor cells in both the
host bone and bone graft [8]. However, it presented some
important adverse effects [3].
PRF, now considered a new generation of platelet con-
centrates [7], has been used in oral and maxillofacial surgery
since 2001 [9]. It consists of an autologous fibrin matrix

13
Vol.:(0123456789)

4574
[10]. In relation to PRP, it presents advantages as it is eas-
ier to prepare and the fact that it does not require chemical
manipulation of the blood, which makes it strictly autolo-
gous [11]. The processes of blood clotting (fibrin polymeri-
zation) and platelet enrichment occur simultaneously in the
preparation of PRF. The coagulation cascade is triggered
when the whole blood comes into contact with the walls of a
silica-coated plastic tube and continues throughout the cen-
trifugation process. This results in the formation of a strong
blood clot from the mechanical point of view (PRF) that
can be handled in surgery. Up to 97% of the total available
platelets can be retained in the preparation of the PRF [11].
The results of different studies [12, 13] reveal that PRF
can be considered a biomaterial with great potential for
healing and bone and soft tissue regeneration as it does not
produce inflammatory reactions and promotes homeostasis,
growth and maturation of bone. Also, in vitro studies showed
that this autologous matrix had a great potential to increase
cell adhesion [14] and to promote proliferation and osteo-
blast differentiation stimuli [15]. Using PRF, Dohan et al.
[11] found immunological and antibacterial properties, lead-
ing to the degranulation of leukocytes and the production of
cytokines with angiogenic and pro/anti-inflammatory func-
tions [16]. One of the most recent advances in PRF technol-
ogy is the production of injectable PRF (i-PRF) [17]. This
biomaterial has several potential applications, however, the
literature on i-PRF and its clinical-biological effects is still
limited [18, 19].
Different to other platelet concentrates, L-PRF is
designed to retain high concentration of leukocytes that are
thought to undergo degranulation during clot formation,
thereby secreting several cytokines that also protect the
wound from infection. Up to 50% of available leukocytes
can be trapped in a platelet concentrate using the L-PRF
preparation method [11]. Besides the role of leukocytes, the
importance of other non-platelet components in the platelet
concentrate remains unclear.
Some authors pointed out the potential antimicrobial
activity of L-PRF [20], but until now, the components
responsible for this activity of L-PRF and the mechanisms
responsible for it remain unclear. Badade et al. [21] studied
the antimicrobial activity of PRP and PRF against bacteria
associated with periodontal disease, such as Aggregatibacter
actinomycetemcomitans and Porphyromonas gingivalis, and
found that PRP was effective in inhibiting these two strains,
but PRF did not showed any inhibition activity. The study
of Castro et al. [20] found that L-PRF membrane inhibited
growth the of Aggregatibacter actinomycetemcomitans (Aa),
Porphyromonas gingivalis (Pg), Fusobacterium nuclea-
tum (Fn) and Prevotella intermedia (Pi), but its greatest
inhibitory effect was observed in Pg. On the other hand,
L-PRF exudate reduced the inhibition growth of Pg in a
dose-dependent manner. The authors did not find inhibitory
13
Molecular Biology Reports (2021) 48:4573–4580
effects against Aa [20]. Recently, Schuldt et al. [22] found
that L-PRF produced a significant decrease in bacterial
count in the biofilm on rough titanium surfaces, probably
through the antimicrobial action of platelets.
Recent studies have reported new advances in platelet
concentrations but in order to validate their advantages com-
paring to conventional L-PRF more long term and controlled
trials are needed. According to Agrawal [23], in clinics, they
should be used with caution.
Taking into account what was mentioned before, the aim
of the present work was to perform a preliminary in vitro
study to analyze the effect of L-PRF on platelet activation,
platelet-leukocytes interactions and antimicrobial activity,
important components in the healing process. The obtained
results would represent a valuable information for additional
research in clinical practice.
Material and methods
The design of the study is shown in Fig. 1. The first step
was the preparation of the L-PRF concentrate according to
a standard protocol approved by FDA and CE (CE646982)
for clinical practice. By compression of the L-PRF, L-PRF
membrane and L-PRF exudate were obtained. For the analy-
ses of antimicrobial activity, L-PRF membrane obtained was
placed in the culture. Molecular biomarkers of platelet acti-
vation and platelet-leukocyte interactions were analyzed by
flow cytometry. As flow cytometry analysis has to be done in
cell suspensions, instead of L-PRF membrane, we added the
L-PRF exudate to autologous whole blood to investigate the
effect of L-PRF on platelet activation and platelet-leukocyte
interactions.
L‑PRF preparation
To prepare the L-PRF, samples of 9 mL of whole blood
were drawn into silica coated plastic vacuum blood collec-
tion tubes without anticoagulant. After collection, the blood
was quickly subjected to centrifugation at 2700 RPM for
12 min (IntraSpin® System—Intra-Lock International—
Boca Raton, FL USA). After the completion of cycle, the
blood in the tube got separated into three distinct layers;
platelet poor plasma at the top, PRF in the middle and a red
blood corpuscular (RBC) base in the bottom. The PRF was
then carefully retrieved from the tube and the RBC base
was carefully removed to retain a small part of it in the clot.
The clot thus obtained was compressed using a Xpression™
Box (IntraSpin® System—Intra-Lock International—Boca
Raton, FL USA) to form L-PRF membrane and L-PRF exu-
date that were used for further analysis (Fig. 1).
Molecular Biology Reports (2021) 48:4573–4580 4575
Fig. 1  Study design: L-PRF
preparation, flow cytometry
analysis of molecular biomarker
in whole blood and antimicro-
bial activity
In vitro platelet activation effect
Blood collection
Blood was collected from 3 healthy donors at the morning
and fasting. These donors did not have any medication in the
previous 10 days. To avoid artefact platelet activation, whole
blood was drawn without a tourniquet from an antecubital
vein and the phlebotomy done with a 19-gauge needle. The
first 2.5 mL was discarded, and 9 mL was collected directly
into a tube containing 1 mL of 129 mM sodium citrate.
Flow cytometry (FCM) analysis of molecular biomarkers
Two samples of each donor were analyzed on a flow Cytom-
eter EPICS XL, Coulter with the set up to measure: forward-
angle light scatter (FALS), right-angle light scatter (90LS),
Fluo-4 and FITC fluorescence (FL1) and PE fluorescence
(FL3).
Intracellular calcium mobilization was measured using
a method described previously by Monteiro et al. [24].
Briefly, 25 µL samples of whole blood preloaded with
Fluo-4 (15 min, at 37 ℃) were incubated with 5 µL of
monoclonal antibody anti-CD41-PE (IMMUNOTECH), to
identify the platelet population, and synthetic tetrapeptide
GPRP was added at 2.5 mM final concentration, to inhibit
fibrin polymerization. After 15 min of incubation at room
temperature in the dark, modified Tyrode’s buffer (1 mL)
was added, and samples of 500 µL were used to determine
kinetic variations of intracellular calcium induced by
L-PRF exudate or α-thrombin (positive control).
The surface expression of platelet receptors activated
GPIIbIIIa and P-selectin were determined by whole-blood
FCM using modified methods previously described [25]
with monoclonal antibodies PAC-1-FITC (Becton Dickin-
son) and the anti-CD62P-FITC (IMMUNOTECH). Briefly,
platelet stimulation was obtained by mixing 25 µL of the
diluted blood sample with 5 µL of GPRP, 5 µL of L-PRF
exudate or 5 µL of α-thrombin solution (positive control).
Then samples were incubated with either anti-CD41-PE
(5  µL) and anti-CD62P-FITC (5  µL) or anti-CD61-PE
(5 µL) and PAC1-FITC (10 µL) for 15 min at room tem-
perature. After this incubation, 1 mL Tyrode’s buffer was
added and samples were analyzed in flow cytometer. In
parallel, 25 µL of unstimulated blood samples were incu-
bated with the labelled antibodies as described above to
assess basal activation.
Antimicrobial activity
In vitro susceptibility of three different American Type Cul-
ture Collection (ATCC) strains was studied with Kirby-Bauer
agar diffusion method: Enterococcus faecalis (ATCC 29212),
Pseudomonas aeruginosa (ATCC 27853) and Candida albi-
cans (ATCC 90028). We choose these strains because of their
clinical relevance. They are the main microorganisms involved
in endodontics and periodontal disease, characterized by rapid
alveolar bone loss and infections of radicular channels, being
13

4576 Molecular Biology Reports (2021) 48:4573–4580

frequently responsible for the failure of the endodontic treat-

ment [26–30]. These microorganisms were also investigated
in a recent study about the inhibitory effects of platelet con-
centrates [31]. The inoculum was prepared with two to three
isolated colonies of each freshly strain to be tested, that were
suspended in 2 mL of sterile saline. The suspensions were
vortexed and cell concentration was adjusted to 0.5 Mc Farland
scale using a densitometer (BioMérieux Marcy-Étoile-France).
A sterile swab was introduced into the inoculum tube and
rotated against the side using firm pressure to remove excess
fluid. The dried surface of Mueller Hinton agar was inocu-
lated by streaking the swab three times over the entire agar
surface and rotate approximately 60 degrees each time to Fig. 2  Alterations of activated GPIIb/IIIa complex (PAC-1) expres-
ensure an even distribution of the inoculum. After 15 min, sion induced by L-PRF. A: Unstimulated platelets. B: Activated
platelets with thrombin 0.5U/ml (positive control) and C: Platelets
to allow the agar plate left to dry, one sterile 6 mm filter incubated with LPRF exudate
paper disk was impregnated with 25 μL of chlorohexidine
0.12% as a positive control and L-PRF circular scalper mem-
brane with a diameter of 6 mm was placed. Each test was
performed in triplicate. Once the tests were placed, inverted
plates were incubated at 35 °C–37 °C for 24 h. Following
incubation, zone sizes inhibition was measured to the nearest
millimeter using a digital caliper made with the unaided eye
while viewing the back of the petri dish. The results of tested
component on 2 samples of each donor were compared with
the positive control.

Ethics statement

Blood samples were collected with the informed consent of
volunteer donors. All procedures performed in this study
involving human participants were in accordance with the
ethical standards of the institutional research committee
and with Helsinki declaration and its later amendments. No
ethical approval was required for this study because human
samples were not identified [32, 33].
Results
In vitro platelet activation effect
L-PRF exudate was used for the in vitro stimulation of
whole blood and analysis of molecular markers of platelet
activation by means of flow cytometry. The main results
from flow cytometry analysis are shown in Figs. 2, 3 and 4.
The surface expression of activated GPIIbIIIa (fibrinogen
receptor) and P-selectin were studied by means of PAC-1
and CD62 antibodies, respectively. Although the values
of platelet activation were different for each donor, all of
them reveal a clear activation response: L-PRF exudate
induced the expression of activated GPIIbIIIa (Fig. 2) and
Fig. 3  Alterations of CD62 expression induced by L-PRF. A: Unstim-
ulated platelets; B: Activated platelets with Thrombin 0.5U/mL (posi-
tive control); and C: Platelets incubated with L-PRF exudate
P-selectin (Fig. 3) in platelets from whole blood in all
samples, in a similar way to thrombin (positive control).
­ a++ mobilization, L-PRF
With respect to intracellular C
exudate also induced strong responses in platelets from
whole blood (Fig. 4). These results were similar for all
samples. Figure 4A shows the C ­ a++ mobilization response
induced by thrombin (positive control) and Fig. 4B shows
the ­Ca++ mobilization induced by the presence of L-PRF.
Antimicrobial activity
To evaluate the antimicrobial activity of platelet concen-
trates we used three strains: Enterococcus faecalis (ATCC
29212), Pseudomonas aeruginosa (ATCC 27853) and Can-
dida albicans (ATCC 90028). Different tests were performed
to assess the antimicrobial activity of L-PRF membrane,
namely Kirby-Bauer agar diffusion method and determina-
tion of the minimum inhibitory concentration.

13
Molecular Biology Reports (2021) 48:4573–4580 4577
Fig. 4  Changes in cytosolic free C ­ a++ in Fluo-4-labeled platelets
stimulated with L-PRF exudate or thrombin. After the determination
of baseline fluorescence of whole blood samples diluted in modified
Tyrode’s buffer, loaded with Fluo-4 (5 µM) and stained with the mon-
The results obtained showed an inhibitory action of
L-PRF membrane for all of microorganisms studied, with
zones of inhibition between 9 and 19 mm, even superior to
the positive control of chlorhexidine 0.12% (between 0 and
12 mm). (Fig. 5).
Discussion
Due to the possibility to delivery growth factors directly to
host tissues, platelet concentrates have been used in differ-
ent areas of medicine, including dentistry, to improve the
healing process [32].
For an adequate repair of the tissues, the collaborative
action of several cells is necessary and the L-PRF repre-
sents a good source of all the elements that contribute to
an adequate healing process. It contains live white blood
cells, trapped in a high-density fibrin network, which also
includes platelets. The immunomodulatory effect of the
presence of leukocytes and certain inflammatory cytokines
[34], the mechanical and biological properties of fibrin, that
contribute to cell migration and tissue regeneration, and the
role of platelets in cell communication, inflammation and
healing responses make L-PRF a potentially important ele-
ment in clinic.
L-PRF is now successfully used as an adjuvant to improve
tissue healing, however there is still a lack of clarification
regarding aspects related to its biological capacity to produce
oclonal antibody specific anti-CD41-PE, L-PRF exudate or thrombin
(0.05 U/mL final concentration) were added at, and changes in green
fluorescence versus time were recorded
and release pro and anti-inflammatory cytokines and growth
factors as well as its contribution to the cell activation [35].
Most of the studies focused on the characterization of
L-PRF content in major growth factors but few studies
focused on its platelet activation effect. Also, more studies
are necessary to confirm its antimicrobial effect although
the antibacterial properties of L-PRF against wound bacteria
were previously described [36]. In a study carried out by
Ciéslik-Bieleckat et al. [31], strains of methicillin-resistant
Staphylococcus aureus (MRSA), methicillin susceptible
Staphylococcus aureus (MSSA), Enterococcus faecalis,
Pseudomonas aeruginosa and Escherichia coli were tested
by the Kirby-Bauer method to two types of thrombin (autolo-
gous and bovine) and to L-PRP. They concluded that L-PRP
was effective against strains MRSA, MSSA, E. faecalis and
P. aeruginosa with inhibition zone diameters between 6 and
18 mm but did not present any antimicrobial effect against E.
coli, and K. pneumoniae. Instead, the addition of thrombin to
L-PRP showed to potentiate the inhibitory action of L-PRP
in susceptible strain [31]. A recent study of Feng et al. [37]
evaluated the inhibitory effect of L-PRF and H-PRF (pre-
pared by horizontal centrifugation) against S. aureus and E.
coli. These authors concluded that H-PRF exhibit a better
antibacterial capacity, what could be attributed to the num-
ber of leukocytes present and the components of the exudate
as PRF solid was less efficient than wet PRF in terms of
antibacterial properties.
13

4578
Fig. 5  Inhibitory effect of
L-PRF on P. aeruginosa, C.
albicans and E. faecalis grow-
ing with antimicrobial diffusion
test performed in agar Mueller–
Hinton A representative cultures
and B quantitative results of
inhibition zone diameters from
three samples
It is important to characterize the profusion of cytokines
and growth factors released, as well as the different mech-
anisms of cell activation and the complex interactions
between them in the L-PRF. Thus, the selection of methods
for preparing platelet-rich products involves issues such as
platelet activation, the number of leukocytes present, platelet
levels, and growth factor enrichment. However, others, such
as ease of use and cost, are also important [38] to consider
L-PRF as a reliable therapeutic option. Different products
of L-PRF can be prepared, namely, L-PRF-membrane and
L-PRF-exudate, being the membrane the most used in chi-
rurgical procedures. Some studies showed that protocols
used impact significantly the cells, growth factors and fibrin
architecture of L-PRF [39]. Also, with respect to antimicro-
bial effect, different results were obtained when using L-PRF
membrane or L-PRF exudate [20, 37].
Taking all this into account, the aim of the present study
was to evaluate the antimicrobial capacity of L-PRF mem-
brane and the in vitro capacity of L-PRF exudate in activat-
ing platelets of whole blood. As far as we know, this is the
first study of whole blood platelet activation using L-PRF
exudate by means of flow cytometry analysis of molecular
biomarkers and also the first one to evaluate the inhibitory
action of L-PRF membrane against three microorganisms
with relevance in oral clinical practice: Pseudomonas aer-
uginosa, Candida albicans and Enterococcus faecalis.
The obtained results clearly show that L-PRF compo-
nents activate platelets from whole blood in a similar way
to thrombin, used here as positive control. Also, L-PRF
13
Molecular Biology Reports (2021) 48:4573–4580
exudate induced the expression of P-selectin in platelets
from whole blood, which is related with platelet-leukocytes
interactions. The third parameter studied, the kinetics of
­Ca2+ mobilization, showed a strong response of whole blood
platelets in the presence of L-PRF exudate. These findings
evidence an important contribution of L-PRF in the heal-
ing process once circulating activated platelets are able to
amplify the effect of activated platelets present in the PRF
and synthesize high levels of several bioactive molecules
with the capacity of stimulating proliferation and activation
of cells involved in wound healing and preventing infection
[40]. The obtained results also reveal that L-PRF membrane
exhibited antimicrobial activity against P. aeruginosa, C.
albicans, and E. faecalis, in agreement what was found in
other studies [31, 41]. Although our results are preliminary
ones, they point to their potential use of L-PRF in the treat-
ment of postoperative infections, namely in the treatment of
periodontal, peri-implant and endodontic infections, refrac-
tory to conventional treatments, that are often produced by
these microorganisms.
An important issue is the preparation of L-PRF, because
at present, there are no data showing how many leukocytes
or platelets within the PRF-based matrices are necessary to
have the best physiological conditions. In this sense, some
studies suggest a partial loss of leukocytes during L-PRF
preparations, pointing out the impact of methodological
variations on the biological products obtained [42]. With
this respect, it is important to highlight that the protocol used
in our study to prepare L-PRF, the IntraSpin system, is the
Molecular Biology Reports (2021) 48:4573–4580 4579
only FDA-cleared medical device for the chairside produc-
tion of Platelet-Rich Fibrin (PRF). Features a protocol that
was scientifically developed and clinically proven from over
200 studies, over 10 + years. This protocol utilizes compo-
nents that are FDA-cleared and CE-marked and have been
optimized to ensure proper material biocompatibility and
clinical performance.
We also think that it is important to point out the use of
L-PRF exudate in addition to membrane in rehabilitation
procedures. According to our results, the use of the L-PRF
exudate could improve treatments by providing a higher
availability of biological factors.
In spite of the limitations of our study, we can conclude
that: (1) the experimental design presented here allows to
evaluate whole blood platelet activation, to analyze molec-
ular biomarkers of other immune cells and particularly to
study platelet-leukocyte interactions; (2) the L-PRF prod-
ucts are efficient in activating platelets of whole blood, and
in inhibiting microbial growth of aerobic and facultative
anaerobic microorganisms that frequently contribute to the
failure of dental treatments, so they can be a valuable tool
in healing process; (3) in our opinion, the use of L-PRF
exudate, in addition to L-PRF membrane, presents some rel-
evant advantages that have to be considered in clinical trials.
Further characterization and quantification of cells and
its products presents in the L-PRF exudate, as well as on the
temporal factor released, is still needed. Also, pre-clinical
and clinical trials will be essential to identify the best activa-
tion and preparation methods for medical and dental applica-
tions of L-PRF. Also, since our results are so promising, in
the future, further studies using strains isolated from clinical
cases will be important.
Acknowledgements  Authors thank the Institute of Research and
Advance Training in Health Sciences and Technologies (IINFACTS-
CESPU) for hosting the study.
Author contributions  All authors have seen and approved the final ver-
sion of the manuscript.
Funding  No funding was received for conducting this study.
Declarations  13. Kim TH, Kim SH, Sándor GK, Kim YD (2014) Comparison of
Conflict of interest  The authors have no relevant financial or non-fi-
nancial interests to disclose.
Ethical approval  Blood samples were collected with the informed con-
sent of volunteer donors. All procedures performed in this study involv-
ing human participants were in accordance with the ethical standards of
the institutional research committee and with Helsinki declaration and
its later amendments. No ethical approval was required for this study
because human samples were not identified.
Informed consent  Each participant signed the corresponding writ-
ten informed consent including the permission to publish the results
obtained in the study.
References
1. Gassling VL, Acil Y, Springer IN, Hubert N, Wiltfang J (2009)
Platelet rich plasma and platelet-rich fibrin in human cell culture.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:48–55.
https://​doi.​org/​10.​1016/j.​tripl​eo.​2009.​02.​007
2. Blair P, Flaumenhaft R (2009) Platelet alpha-granules: basic biol-
ogy and clinical correlates. Blood Rev 23:177–189. https://​doi.​
org/​10.​1016/j.​blre.​2009.​04.​001
3. Kiran NK, Mukunda KS, Tilak Raj TN (2011) Platelet concen-
trates: a promising innovation in dentistry. J Dent Sci Res 2:50–61
4. Yeaman MR (1997) The role of platelets in antimicrobial host
defense. Clin Infect Dis 25:951–970. https://​doi.​org/​10.​1086/​
516120
5. Krijgsveld J, Zaat SA, Meeldijk J, Van Veelen PA, Fang G,
Poolman B, Brandt E, Ehlert JE, Kuijpers AJ, Engbers GH, Fei-
jen J et al (2000) Thrombocidins, microbicidal proteins from
human blood platelets, are C-terminal deletion products of CXC
chemokines. J Biol Chem 275:20374–20381. https://​doi.​org/​10.​
1074/​jbc.​275.​27.​20374
6. Jenne CN, Kubes P (2015) Platelets in inflammation and infec-
tion. Platelets 26:286–292. https://​doi.​org/​10.​3109/​09537​104.​
2015.​10104​41
7. Borie E, Olivi DG, Orsi IA, Garlet K, Weber B, Beltran V, Fuentes
R (2005) Platelet rich fibrin application in dentistry: a literature
review. Int J Clin Exp Med 8(5):7922–7929
8. Schmitz J, Hollinger J (2001) The biology of platelet-rich plasma.
J Oral Maxillofac Surg 59(9):1119–1121. https://d​ oi.o​ rg/1​ 0.1​ 053/​
joms.​2001.​2680
9. Choukroun J, Adda F, Schoeffler C, Vervelle A (2001) Une oppor-
tunité en paro-implantologie: Ie PRF. Implantodontie 42:55–62
10 DohanEhrenfest DM, Choukroun J, Diss A, Dohan S, Dohan AJ,
Mouhyi J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-
generation platelet concentrate. Part I: technological concepts and
evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
101(3):e37–e44. https://​doi.​org/​10.​1016/j.​tripl​eo.​2005.​07.​008
11 DohanEhrenfest DM, Del Corso M, Diss A, Mouhyi J, Charrier JB
(2010) Three-dimensional architecture and cell composition of a
Choukroun’s platelet-rich fibrin clot and membrane. J Periodontol
81(4):546–555. https://​doi.​org/​10.​1902/​jop.​2009.​090531
12. Joseph VR, Sam G, Amol NV (2014) Clinical evaluation of autol-
ogous platelet rich fibrin in horizontal alveolar bony defects. J
Clin Diagn Res 8(11):ZC43–ZC47. https://d​ oi.o​ rg/1​ 0.7​ 860/J​ CDR/​
2014/​9948.​5129
platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and concen-
trated growth factor (CGF) in rabbit-skull defect healing. Arch Oral
Biol 59(5):550–558. https://​doi.​org/​10.​1016/j.​archo​ralbio.​2014.​02.​
004
14. Wu CL, Lee SS, Tsai CH, Lu KH, Zhao JH, Chang YC (2012)
Platelet-rich fibrin increases cell attachment, proliferation and col-
lagen-related protein expression of human osteoblasts. Aust Dent J
57:207–212. https://​doi.​org/​10.​4103/​0976-​237X.​142830
15. DohanEhrenfest DM, Diss A, Odin G, Doglioli P, Hippolyte MP,
Charrier JB (2009) In vitro effects of Choukroun´s PRF (platelet-
rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes,
preadipocytes, and maxillofacial osteoblasts in primary cultures.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108(3):341–352.
https://​doi.​org/​10.​1016/j.​tripl​eo.​2009.​04.​020
13

4580
16 DohanEhrenfest DM, Choukroun J, Diss A, Dohan S, Dohan AJ,
Mouhyi J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-
generation platelet concentrate. Part III: technological concepts
and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
101(3):e51–e55. https://​doi.​org/​10.​1016/j.​tripl​eo.​2005.​07.​010
17. Mourão CF, Valiense H, Melo ER, Mourão NB, Maia MD (2015)
Obtention of injectable platelets rich-fibrin (i-PRF) and its polymeri-
zation with bone graft: technical note. Rev Col Bras Cir 42(6):421–
423. https://​doi.​org/​10.​1590/​0100-​69912​01500​6013
18 Jain NK, Gulati M (2016) Platelet-rich plasma: a healing virtuoso.
Blood Res 51(1):3–5. https://​doi.​org/​10.​5045/​br.​2016.​51.1.3
19 Maria-Angeliki G, Alexandros-Efstratios K, Dimitris R, Konstanti-
nos K (2015) Platelet-rich plasma as a potential treatment for nonci-
catricial alopecias. Int J Trichol 7(2):54–63. https://​doi.​org/​10.​4103/​
0974-​7753.​160098
20. Castro AB, Herrero ER, Slomka V, Pinto N, Teughels W, Quirynen
M (2019) Antimicrobial capacity of leukocyte-and platelet rich
fibrin against periodontal pathogens. Sci Rep 9:8188. https://​doi.​
org/​10.​1038/​s41598-​019-​44755-6
21. Badade PS, Mahale SA, Panjwani AA, Vaidya PD, Warang AD
(2016) Antimicrobial effect of platelet-rich plasma and platelet-rich
fibrin. Indian J Dent Res 27:300–304
22. Schuldt L, Bi J, Owen G, Shen Y, Haapasolo M, Hakkinen L, Ler-
java H (2020) Decontamination of rough implant surfaces colonized
by multispecies oral biofilm by application of leukocyte- and plate-
let-rich fibrin. J Periodontol. https://​doi.​org/​10.​1002/​JPER.​20-​0205
(Online ahead of print)
23 Agrawal AA (2017) Evolution, current status and advances in appli-
cation of platelet concentrate in periodontics and implantology.
World J Clin Cases 5(5):159–171. https://​doi.​org/​10.​12998/​wjcc.​
v5.​i5.​159
24. Monteiro MC, Sansonetty F, Gonçalves MJ, O’Connor JE (1999)
Flow cytometric kinetic assay of calcium mobilization in whole
blood platelets using Fluo-3 and CD41. Cytometry 35(4):302–310
25. Schmitz G, Rothe G, Ruf A, Barlage S, Tschöpe D, Clemetson
KJ, Goodall AH, Michelson AD, Nurden AT, Shankey TV (1998)
Review European working group on clinical cell analysis: consensus
protocol for the flow cytometric characterisation of platelet function.
Thromb Haemost 79(5):885–896
26. Siqueira JF, Rôças IN, Silva MG (2008) Prevalence and clonal analy-
sis of Porphyromonas gingivalis in primary endodontic infections.
J Endod 34(11):1332–1336. https://​doi.​org/​10.​1016/j.​joen.​2008.​08.​
021
27. Lima SM, Sousa MG, Freire M de S, de Almeida JA, Cantuária
AP, Silva TA, de Freitas CG, Dias SC, Franco OL, Rezende TM
(2015) Immune response profile against persistent endodontic patho-
gens candida albicans and enterococcus faecalis in vitro. J Endod
41(7):1061–5. https://​doi.​org/​10.​1016/j.​joen.​2015.​02.​016
28. Siqueira JF Jr, Rôças IN (2004) Polymerase chain reaction-based
analysis of microorganisms associated with failed endodontic
treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
97(1):85–94. https://​doi.​org/​10.​1016/​s1079-​2104(03)​00353-6
(PMID: 14716262)
29. Souto R, Colombo APV (2008) Prevalence of Enterococcus faeca-
lis in subgingival biofilm and saliva of subjects with chronic peri-
odontal infection. Arch Oral Biol 53(2):155–160. https://​doi.​org/​10.​
1016/j.​archo​ralbio.​2007.​08.​004
30. Lee L-W, Lee Y-L, Hsiao S-H, Lin H-P (2017) Bacteria in the apical
root canals of teeth with apical periodontitis. J Formos Med Assoc
116(6):448–456. https://​doi.​org/​10.​1016/j.​jfma.​2016.​08.​010
13
Molecular Biology Reports (2021) 48:4573–4580
31. Cieslik-Bielecka A, Bold T, Ziolkowski G, Pierchala M, Kro-
likowska A, Reichert P (2018) Antibacterial activity of leukocyte-
and platelet-rich plasma: an in vitro study. Biomed Res Int 2018:8.
https://​doi.​org/​10.​1155/​2018/​94717​23 (Article ID 9471723)
32 Miron RJ, Fujioka-Kobayashi M, Hernandez M, Kandalam U, Zhang
Y, Ghanaati S, Choukroun J (2017) Injectable platelet rich fibrin
(i-PRF): opportunities in regenerative dentistry? Clin Oral Investig
21(8):2619–2627. https://​doi.​org/​10.​1007/​s00784-​017-​2063-9
33. Fujioka-Kobayashi M, Schaller B, Morão CF, Zhang Y, Sculean
A, Miron RJ (2020) Biological characterization of an injectable
platelet-rich fibrin mixure consisting of autologous albumin gel and
liquid platelet-rich-fibrin (Alb-PRF). Platelets 20:1–8. https://​doi.​
org/​10.​1080/​09537​104.​2020.​17174​55
34. Van Dooren FH, Duijvis NW, te Velde AA (2013) Analysis of
citokines and chemokines produced by whole blood, peripheral
mononuclear and polymorphonuclear cells. J Immunol Methods
396(1–2):128–133. https://​doi.​org/​10.​1016/j.​jim.​2013.​08.​006
35. Jiménez-Aristizabal RF, López C, Álvarez ME, Giraldo C, Prades
M, Carmona JU (2017) Long-term cytokine and growth factor
release from equine platelet-rich fibrin clots obtained with two dif-
ferent centrifugation protocols. Cytokine 97:149–155. https://​doi.​
org/​10.​1016/j.​cyto.​2017.​06.​011
36. Edelblute CM, Donate AL, Hargrave BY, Heller IC (2015) Human
platelet gel supernatant inactivates opprtunistic wound pathogens
on skin. Platelets 6:13–16. https://​doi.​org/​10.​3109/​09537​104.​2013.​
863859
37. Feng M, Wang Y, Zhang P, Zhao Q, Yu S, Shen K, Miron RJ, Zhang
Y (2020) Antibacterial effects of platelet-rich fibrin produced by
horizontal centrifugation. Int J Oral Sci 12:32. https://​doi.​org/​10.​
1038/​s41368-​020-​00099-w
38 Davis V, Abukabda AB, Radio NM, Witt-Enderby PA, Clafshen-
kel WP, Cairone J, Rutkowski J (2014) Platelet rich preparation
to improve healing. Part II: platelet activation and enrichment,
leukocyte inclusion, and other selection criteria. J Oral Implantol
4:511–521. https://​doi.​org/​10.​1563/​AAID-​JOI-D-​12-​00106
39. Choukroun J, Ghanaati S (2018) Reduction of relative centrifuga-
tion force within injectable platelet-rich-fibrin (PRF) concentrates
advances patients’ own inflammatory cells, platelets and growth fac-
tors: the first introduction to the low speed centrifugation concept.
Eur J Trauma Emerg Surg 44(1):87–95. https://​doi.​org/​10.​1007/​
s00068-​017-​0767-9
40. Toffler M (2014) Guided bone regeneration using cortical bone pins
in combination with leukocyte-and-platelet-rich fibrin (L-PRF).
Compend Contin Educ Dent 35(2):192–198
41 D’asta F, Halstead F, Harrison P, ZecchiOrlandini S, Moiemen N,
Lord J (2018) The contribution of leukocytes to the antimicrobial
activity of platelet-rich plasma preparations: a systematic review.
Platelets 29(1):9–20. https://​doi.​org/​10.​1080/​09537​104.​2017.​13177​
31
42. de Oliveira LA, Soares RO, Buzzi M, Mourão CFAB, Kawase T,
Kuckelhaus SAS (2020) Cell and platelet composition assays by
flow cytometry: basis for new platelet-rich fibrin methodologies. J
Biol Regul Homeost Agents 34(4):1379–1390. https://​doi.​org/​10.​
23812/​20-​278-A
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.