Platelets

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Platelet rich fibrin as a bioactive matrix with

proosteogenic and proangiogenic properties on
human healthy primary cells in vitro

Eva Dohle, Lena Schmeinck, Kamelia Parkhoo, Robert Sader & Shahram
Ghanaati

To cite this article: Eva Dohle, Lena Schmeinck, Kamelia Parkhoo, Robert Sader &
Shahram Ghanaati (2024) Platelet rich fibrin as a bioactive matrix with proosteogenic and
proangiogenic properties on human healthy primary cells in vitro, Platelets, 35:1, 2316744,
DOI: 10.1080/09537104.2024.2316744

To link to this article: https://doi.org/10.1080/09537104.2024.2316744

© 2024 The Author(s). Published with

license by Taylor & Francis Group, LLC.

Published online: 23 Feb 2024.

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ISSN: 0953-7104 (print), 1369-1635 (electronic)

Platelets, 2024; 35(1): 2316744

© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
DOI: https://doi.org/10.1080/09537104.2024.2316744

RESEARCH ARTICLE

Platelet rich fibrin as a bioactive matrix with proosteogenic and

proangiogenic properties on human healthy primary cells in vitro
Eva Dohle*, Lena Schmeinck*, Kamelia Parkhoo, Robert Sader, & Shahram Ghanaati

FORM, Frankfurt Orofacial Regenerative Medicine, Department for Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Medical Center of the Johann
Wolfgang Goethe University, Frankfurt, Germany

Abstract Keywords
Angiogenesis, bone tissue regeneration, low-
Blood concentrates like platelet rich fibrin (PRF) have been established as a potential auto­
speed centrifugation concept, osteogenic dif­
logous source of cells and growth factors with regenerative properties in the field of dentistry ferentiation, platelet rich fibrin
and regenerative medicine. To further analyze the effect of PRF on bone tissue regeneration,
this study investigated the influence of liquid PRF matrices on human healthy primary History
osteoblasts (pOB) and co-cultures composed of pOB and human dermal vascular endothelial
Received 6 December 2023
cells (HDMEC) as in vitro model for bone tissue regeneration. Special attention was paid to
Revised 2 January 2024
the PRF mediated influence on osteoblastic differentiation and angiogenesis. Based on the
Accepted 4 January 2024
low-speed centrifugation concept, cells were treated indirectly with PRF prepared with a low
(44 g) and high relative centrifugal force (710 g) before the PRF mediated effect on osteoblast
proliferation and differentiation was assessed via gene and protein expression analyses and
immunofluorescence. The results revealed a PRF-mediated positive effect on osteogenic
proliferation and differentiation accompanied by increased concentration of osteogenic
growth factors and upregulated expression of osteogenic differentiation factors.
Furthermore, it could be shown that PRF treatment resulted in an increased formation of
angiogenic structures in a bone tissue mimic co-culture of endothelial cells and osteoblasts
induced by the PRF mediated increased release of proangiogenic growth factors. The effects
on osteogenic proliferation, differentiation and vascularization were more evident when low
RCF PRF was applied to the cells. In conclusion, PRF possess proosteogenic, potentially
osteoconductive as well as proangiogenic properties, making it a beneficial tool for bone
tissue regeneration.

Plain Language Summary

What is the context?
The treatment of bone defects is still a challenge in the field of regenerative medicine. In this
context, researchers and clinicians are continuously focusing on developing new therapeutic
strategies like the use of autologous blood concentrates like Platelet rich fibrin (PRF) to
improve regeneration by directly delivering wound healing promoting cells and growth factors
to the defect side in order to restore the structure and functional integrity of damaged hard
tissue in combination with adequate tissue regeneration.
What is new?
Focus of the present in vitro study was to further evaluate the potential of PRF paying particular
attention to the PRF-mediated effect on osteogenic differentiation and angiogenesis of human
primary osteoblasts as well as on a more complex tissue like co-culture consisting of osteoblasts
and microvascular endothelial cells. We could demonstrate that PRF is able to support and affect
a variety of processes involved in bone tissue regeneration including osteogenic proliferation,
osteogenic differentiation as well as angiogenic structure formation.

*These authors contributed equally to this work.

Correspondence: Eva Dohle, FORM (Frankfurt Orofacial Regenerative

Medicine),
Department for Oral, Cranio-Maxillofacial and Facial Plastic Surgery,
Medical Center of the Johann Wolfgang Goethe University, Theodor-
Stern-Kai 7, Frankfurt 60590, Germany. E-mail: dohle@med.uni-frank­
furt.de
This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/
4.0/), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The terms on
which this article has been published allow the posting of the Accepted
Manuscript in a repository by the author(s) or with their consent.
2 E. Dohle et al. Platelets, 2024; 35(1): 1–11

Treatment of PRF resulted in:

- increased cell viability*
- higher expression of osteogenic differentiation factors*
- higher release of osteogenic growth factors*
- increased formation of microvessel-like structures*
(*compared to untreated control)
What is the impact?
PRF represents a beneficial autologous tool for regenerative purposes combining proosteo­
genic and proangiogenic properties. Therefore, PRF might be used for applications in versatile
fields of medicine in the context of improving bone tissue regeneration.

Introduction
Tissue regeneration is of crucial importance in medical interventions
and in the restoration of health, thereby being of great significance in
all medical fields. Consequently, there is a need to therapeutically
increase and control the process of regeneration to minimize com­
plications and to maintain the patient’s comfort while achieving an
optimal and long-term clinical outcome.1,2 A particular challenge in
the field of regenerative medicine is the treatment of bone defects
since autologous bone transplants are associated with major risks
such as infections, pain, hematoma, nerve damage, and scarring.
Therefore, clinicians are continuously focusing on finding alterna­
tives to restore the structure and functional integrity of damaged hard
tissue in combination with adequate tissue regeneration.3,4 While the
interaction of immune cells, the assembly of extracellular matrix
components and the release of growth factors and cytokines have
a significant impact on the process of wound healing and regenera­
tion process, researchers have started to develop new therapeutic
strategies like the use of autologous blood concentrates improving
regeneration by directly delivering wound healing promoting cells
and growth factors to the defect side. In recent years, a variety of
blood concentrates such as platelet rich plasma and platelet rich
fibrin gained significant attention in the field of dentistry and regen­
erative medicine.5–8 Platelet rich fibrin is a second-generation blood
concentrate obtained by collecting and centrifuging the patient´s own
blood that has been established as a convenient tool for a wide range
of medical and dental indications such as guided bone regeneration,
implantation and regeneration of periodontal intrabody defects.9,10 In
addition, it has already been shown that PRF can be widely utilized
for the biologization of biomaterials such as bone substitute materi­
als and collagen membranes to trigger a material-induced tissue
healing response.11–13 In this regard, clinical trials have revealed
that PRF treatment reduces pain development and improves tissue
regeneration while minimizing the occurrence of complications such
as scarring and inflammation.13–15 Several in vitro studies regarding
the composition of PRF identified PRF as a natural, endogenous
source of high concentrations of growth factors, cytokines, fibrin and
cells such as leukocytes, platelets and stem cells.10,16,17 With respect
to the cell content and growth factor release, previous studies have
revealed that the relative centrifugation force (RCF) affect the com­
position of PRF. Since it could be shown that a higher concentration
of cells and growth factors could be observed when the RCF was
reduced, the low-speed centrifugation concept (LSCC) was
introduced.18 Furthermore, by providing multiple growth factors
such as Platelet derived growth factor (PDGF), Transforming growth
factor ß (TGF-β), epidermal growth factor (EGF) and Vascular
endothelial growth factor (VEGF), PRF has been found to have
a positive impact on wound healing and regeneration associated
processes.17,19,20 Although a number of studies are dealing with
the evaluation of the effect of PRF in vitro, there is little recent
evidence regarding the effects of indirectly applied PRF on the
proliferation and differentiation capacity of human healthy osteo­
blasts or more complex in vitro human bone tissue mimics. During
this study, the potential PRF mediated effect on human healthy
primary osteoblasts (pOBs) has been evaluated with regard to cell
proliferation, osteogenic differentiation, growth factor content and
gene expression analyses of osteogenic differentiation factors. In
addition, since a better understanding of bone healing mechanisms
can be generally assisted by more complex tissue-like constructs,21
the effect of PRF was also analyzed in an innovative in vitro co-
culture model consisting of primary osteoblasts and primary
endothelial cells with regard to angiogenesis and angiogenesis asso­
ciated factors.
Materials and methods
Ethical statement
All cells that were used for this study were obtained from excess tissue
and their application was in accordance with the principle of informed
consent and approved by the responsible Ethics Commission of the
state Hessen, Germany. In addition, the application of PRF in this
study was approved by the responsible Ethics Commission of the state
of Hessen, Germany (265/17) and all donors gave informed consent to
the use of their blood for study purposes.
Primary cells
Human primary osteoblasts (pOB) were obtained from healthy
cranial bone obtained as excess tissue during surgery. Cells were
isolated from bone fragments according to an established
protocol22 and were cultivated in Dulbecco´s Modified Eagle´s
Medium F-12 Ham (Sigma-Aldrich, St. Louis, USA) supplemented
with 10% FCS and 1% penicillin/streptomycin (Sigma-Aldrich,
St. Louis, USA) at 37°C in a humidified atmosphere. Primary
osteoblasts were utilized up to passage 5. Human dermal micro­
vascular endothelial cells were isolated from healthy tissue rem­
nants obtained during lip operations and according to an
established protocol.23 HDMECs were cultivated in T-25 cell cul­
ture flasks previously coated with gelatin (0.02%) in Endothelial
Cell Growth Medium MV containing 1% penicillin/streptomycin.

Table I. Centrifugation protocols according to the low speed centrifugation concept (LSCC). In all experimental set-ups, the
high (710 ×g) and the low (44 ×g) centrifugation protocols were selected for comparison.

RPM time g-force Tube material

Low relative centrifugal force (low RCF) 600 rpm 8 min. 44 g plastic
High relative centrifugal force (high RCF) 2400 rpm 8 min. 710 g plastic
DOI: https://doi.org/10.1080/09537104.2024.2316744
Preparation of PRF
Whole blood was collected from at least 3 donors of all genders
who gave informed consent to participate in the study. The donors
were healthy and free of infectious diseases, did not take antic­
oagulants, nor did they consume alcohol or nicotine. Their ages
ranged from 20 to 50 years. For PRF preparation, 10 ml of periph­
eral blood was drawn from the median cubital vein and collected
into plastic-coated PRF tubes (Process for SYBR®, Nice, France).
The blood was immediately centrifuged in a Duo centrifuge
(Cologne, Mectron) with a 110 mm radius fixed angle rotor.
Based on the LSCC, the high RCF PRF protocol and the low
RCF PRF protocol were implemented in our studies (Table I).
After centrifugation, the top clear, yellow-colored layer of liquid
PRF was collected using a syringe individually for each donor in
sterile 15 ml tubes and then homogenized and the still liquid PRF
was used for cell culture experiments.
Cultivation of primary osteoblasts with PRF
For indirect PRF application, 100.000 human pOBs per well were
pre-seeded in 24-well plates and cultivated in DMEM F-12 Ham
containing 10% FCS and 1% penicillin/streptomycin for 24 h. For
the separation of PRF and the pre-seeded cells, trans-well inserts
with a pore size of 0.4 μm were utilized allowing only growth
factors and signaling molecules to pass through the membrane,
but not the cells. Therefore, the trans-wells were placed in the pre-
seeded 24-well plates and either 100 μL of low RCF PRF or 100
μL of high RCF PRF was added directly into the trans-wells. Wells
containing solely human pOBs without the addition of PRF served
as controls. After the clotting process of PRF was completed, 1 ml
of DMEM F-12 Ham containing 10% FCS and 1% penicillin/
streptomycin was added to the lower compartment of the wells.
An additional 100 μl of medium was added to the inner part of each
trans-well to prevent the PRF from drying. Cells and cell/PRF
complexes were cultivated for 9 days. Supernatants were collected
after 2, 3, 7 and 9 days and stored for ELISA. Finally, the cells
were analyzed for cell viability after 3, 7 and 9 days as well as fixed
for immunofluorescence staining after 2 days and processed for
gene expression analyses after 7 days of PRF treatment.
Co-culture experimentation
10.000 human pOBs/well and 10.000 HDMECs/well were mixed
and co-seeded on thermanox™ cell culture coverslips (Karlsruhe,
Thermo Fisher Scientific) in 24-well plates cultivated in
Endothelial Cell Growth Medium MV with 1% penicillin/strepto­
mycin. After 24 h, a trans-well filter system with a pore structure
of 0.4 μm was inserted into the wells. 100 μl of PRF was added to
each trans-well. Co-culture/PRF complexes as well as controls
without PRF were cultured for 7 and 14 days. Supernatants were
collected and stored for ELISA after 4, 7, 10 and 14 days. In
addition, cells were fixed for immunofluorescence staining after 7
and 14 days of cultivation.
Cell viability assay
The effect of indirect PRF treatment on cell viability of pOBs was
examined after 3, 7 and 9 days of cultivation. After washing the
wells with PBS, DMEM F-12 Ham containing 10% FCS and 1%
penicillin/streptomycin and 100 µl CellTiter 96ⓇAQueous One
Solution Reagent (Promega, Madison, USA) were added followed
by an incubation period of 1 h at 37°C. 100 μl from each well was
transferred to a separate 96-well plate and absorbance was mea­
sured at 490 nm in a microplate reader (GENios plus, TECAN,
Crailsheim, Germany).
proosteogenic and proangiogenic properties of PRF 3
Immunofluorescence staining
For immunofluorescence staining cells were fixed in 4% buffered
formalin (Roti-Histofix 4%, acid-free pH7, Carl-Roth, Germany)
washed 3 times with phosphate-buffered saline (PBS), permeabi­
lized for intracellular antigens with 0.1% Triton-X100/PBS and
incubated with the specific primary antibody (mouse alpha-
smooth muscle actin) diluted in 1% BSA/PBS solution (1:2000,
Dako), for 1 h at RT (room temperature). Fluorescently labeled
secondary antibodies (Alexa fluor 488 anti-mouse) were diluted in
1% BSA/PBS and incubated in a dark environment for 1 h at RT.
Thereafter, the cell nuclei were counterstained with DAPI diluted in
1% BSA/PBS (1:1000). The fluorescent D-LEDI LED illumination
system of the Eclipse Ni/E with the Nikon DS-Ri2 camera (Nikon
eclipse Ni/E, Düsseldorf, Germany) was used for evaluation.
Image quantification
Three randomly chosen immunofluorescently stained (CD31)
images from each experimental group of the co-culture experi­
ments were examined at 20-fold magnification for the presence of
CD31-positive stained vascular structures. The Eclipse Ni/E
fluorescence microscope with a DS-Ri2 camera (Nikon,
Düsseldorf, Germany) was used to acquire the images. When
quantifying the images in Nikon´s NIS-elements program
(Nikon, Düsseldorf, Germany), CD31-positive stained vascular
structures were manually selected with the measurement tool
and total length of vascular structures in pixels were exported to
MS Excel. The values were calculated in percentage by setting the
control cultures to 100% as a reference value. Samples without
the addition of PRF served as controls.
Enzyme-linked immunosorbent assay (ELISA)
The collected cell culture supernatants were quantified for relative
growth factor concentrations of TGF-β, PDGF-BB and VEGF
using ELISA-DuoSet development system (R&D Systems)
according to the manufacturer´s instructions. A microplate reader
(Infinite M200, Tecan, Crailsheim, Germany) detected the optical
density of each well at a wavelength of 450 nm.
RNA isolation and gene expression analyses
RNA isolation was performed using the RNeasy Micro Kit (Qiagen,
Hilden, Germany) according to the manufacturer´s instructions.
Using the standard protocol of Qiagen´s Omniscript reverse tran­
scription kit, 1 μg of the extracted RNA per sample was transcribed
into complementary DNA (cDNA). The relative gene expression of
osteogenic differentiation factors was analyzed using specific pri­
mers for BMP-1, BMP-2, col1, osteonectin (ON), osteopontin
(OPN) and alkaline phosphatase (ALP). For quantitative reverse
transcription polymerase chain reaction (qRT-PCR), the RT-PCR
cycler StepOnePlus by Applied Biosystems was implemented.
SYBR® green was utilized as a DNA-binding fluorescent dye.
Analyses were performed in triplicates with a standardized cycler
program: 94°C for 2 minutes, 94°C for 15 s and 60°C for 1 minute.
The reactions were run for 40 cycles. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) served as the endogenous standard. The
relative gene expression was determined using the ∆∆Ct method.
As a reference value, the control cultures were set to one.
Statistical evaluation
All experiments were performed with at least three different
donors. The data presented were evaluated as mean ± standard
deviation (SD). Statistical significance was calculated with the one-
way multifactorial variance analysis ANOVA test or t-test using
4 E. Dohle et al.
graphed pad prism 9 (GraphPad Software Inc.). Statistical signifi­
cance was assessed when * p < 0.05, ** p < 0.01, *** p < 0.001
and **** p < 0.0001 and documented in the figures.
Results
PRF-mediated effect on cell morphology and cell viability of
pOBs
Indirect application of PRF aimed to investigate the PRF
mediated effect on the morphology and proliferation of pOBs.
In this regard, monocultured pOBs were stained for smooth mus­
cle actin antibody (SMA) by immunofluorescent staining and
counterstained with 4′,6-Diamidino-2-phenylindole (DAPI)
(Figure 1A). In general, a higher cell amount could be observed
in pOBs treated with PRF compared to pOBs without indirect
PRF treatment. Primary osteoblasts treated with low RCF PRF
formed a confluent cell layer covering the entire bottom of the
cell culture well. In low RCF PRF treated samples, the cell
number was observed to be higher than in pOBs treated with
high RCF PRF. In pOBs without PRF treatment and pOBs treated
with high RCF PRF, the cell layer was found to be semiconfluent
and cell distribution and cell arrangement resembled a structure
with gaps. In addition, the determination of the relative number of
viable cells revealed a significant higher amount of pOBs when
treated indirectly with PRF compared to pOBs without PRF
treatment at all time points (Figure 1C). When measuring the
total RNA concentration isolated from monocultured pOBs with
and without PRF treatment after 2 days of cultivation,
a statistically significant higher amount of total RNA could be
detected in samples cultivated with PRF compared to pOBs with­
out PRF treatment. In this regard, the amount of isolated RNA in
Figure 1. A. Analysis of the morphology of monocultured pOB with and without PRF treatment after 2 days. POBs were stained for SMA (green) and
DAPI (blue) by immunofluorescence staining. B. Isolation of RNA from monocultured pOBs treated with PRF compared to untreated pOBs after 2
days. C. Effect of indirectly applied PRF on the viability of pOBs seeded in monoculture after 3, 7 and 9 days. Statistical significance was assessed by
****p < 0.0001. Scale bars: 500 μm.
Platelets, 2024; 35(1): 1–11
samples of pOBs treated with low RCF PRF tended to be higher
compared to pOBs treated with high RCF PRF (Figure 1B).
Influence of PRF on osteogenic differentiation of pOB
For evaluation of the influence of PRF on osteogenic differentia­
tion, gene expression levels of osteogenic differentiation factors
(ALP, col-1, SPARC, SPP1, BMP-1, BMP-2) were assessed in
samples of pOBs with and without indirect PRF treatment for 7
days (Figure 2A). A higher expression of ALP (alkaline phospha­
tase), col-1 (collagen type 1), BMP-1 (bone morphogenic pro­
tein 1), BMP-2 and SPARC (osteonectin) could be determined in
PRF treated pOBs compared to untreated pOBs. Primary osteo­
blasts treated with low RCF PRF appeared to express higher
levels of col-1, BMP-1, BMP-2 and SPARC compared to pOBs
treated with high RCF PRF. Evaluation of the SPP1 (osteopontin)
gene expression was found to be downregulated when pOBs were
combined with PRF compared to untreated pOBs.
Determination of osteogenic growth factors in pOBs in
response to PRF
The protein concentration of the osteogenic growth factors TGF-
β1 and PDGF-BB was determined after 3, 7 and 9 days in super­
natants of indirect PRF treated pOBs compared to controls
(Figure 3). On day 3, TGF-β1 levels did not differ significantly
when supernatants of untreated pOBs were compared to super­
natants of pOBs treated with PRF. On the 7th day, supernatants
of pOBs treated with low RCF PRF contained statistically sig­
nificant higher TGF-β1 levels compared to the other experimen­
tal groups, while TGF-β1 values of untreated pOB and pOB
treated with high RCF PRF hardly differed. After a cultivation
DOI: https://doi.org/10.1080/09537104.2024.2316744 proosteogenic and proangiogenic properties of PRF 5

Figure 2. Determination of the relative gene expression of osteogenic differentiation factors in cultures of pOBs with and without indirect PRF
treatment using specific primers for alkaline phosphatase (ALP), collagen type 1 (col-1), osteonectin (SPARC), osteopontin (SPP1), BMP-1 and BMP-
2 by real time PCR after a 2-day incubation period.

Figure 3. Evaluation of the release of the proosteogenic growth factors TGF-β1 and PDGF-BB from cell culture supernatants of monocultured pOB
after 3, 7 and 9 days. Statistical significance was calculated using ANOVA and was assessed by **p < 0.01, ***p < 0.001 and ****p < 0.0001.
6 E. Dohle et al. Platelets, 2024; 35(1): 1–11

period of 9 days, TGF-β1 concentrations in supernatants of
pOBs treated with low and high RCF PRF were statistically
significant higher compared to supernatants of untreated pOB.
In this regard, low RCF PRF treated pOBs contained statistically
significant higher levels of TGF-β1 than supernatants of pOBs
treated with high RCF PRF. The cumulative TGF-β1 concentra­
tion increased in all experimental groups during the course of
cultivation from the 3rd to the 7th day. PDGF-BB concentration
in supernatants of pOBs treated with low RCF PRF was statis­
tically significant higher compared to supernatants of the other
experimental groups at all time points. In this regard, the highest
PDGF-BB concentrations could be measured after 3 days of
cultivation. After a cultivation period of 7 and 9 days, the
detectable PDGF concentrations decreased. While supernatants
of pOBs treated with high RCF PRF contained low levels of
PDGF-BB on day 3, PDGF-BB values could not be measured in
supernatants at any of the other time points. In supernatants of
monocultured pOBs without PRF treatment, PDGF-BB was also
not detectable at any tested time point.
Analysis of microvessel-like structure formation of HDMECs
in co-culture with pOBs in response to PRF
Co-culturing of human dermal microvascular endothelial cells
(HDMECs) and primary osteoblasts (pOBs) followed by indirect
PRF treatment aimed to investigate the effect of PRF on vascular

Figure 4. Analysis of microvessel-like structure formation in an in vitro co-culture model for bone tissue engineering. A. Immunofluorescence staining
for the endothelial cell type specific marker CD31 of co-cultures consisting of HDMEC and pOB. Co-cultures were either treated with PRF or without
PRF (control group) for 7 (upper row) and 14 (lower row) days. B. Evaluation of the relative amount of vascular structure formation after 7 and 14
days. Statistical significance was assessed by **p < 0.01 and ****p < 0.0001. Scale bars 150 μm.
DOI: https://doi.org/10.1080/09537104.2024.2316744
structure formation after 7 and 14 days. The activation of endothe­
lial cells within the co-culture system for angiogenic structure
formation was visualized by immunofluorescent staining for the
endothelial marker PECAM-1 (CD31) (Figure 4A). In co-culture
systems without the addition of PRF (control group) and with the
addition of high RCF PRF, the typical cobblestone-like morphol­
ogy of aggregated HDMECs without the formation of vascular
structures became visible after 7 days. In comparison, co-cultures
cultivated with low RCF-PRF revealed the formation of vessel
structures after 7 days of cultivation. After a cultivation period of
14 days, the HDMEC aggregates started to sprout out and exhib­
ited an increasing formation of microvessel-like structures in all
experimental groups. While co-cultures treated with low RCF
PRF revealed the highest increase in vascular structure formation,
the amount of microvessel-like structure formation was lower in
co-cultures treated with high RCF PRF and co-cultures without
PRF treatment. The quantitative evaluation of vascular structure
formation confirmed the results (Figure 4B). PRF treatment
resulted in an increased percentage of vascular structure forma­
tion and induced a more complex network of angiogenic struc­
tures compared to co-cultures without PRF treatment after 7 and
14 days. In this regard, the addition of low RCF PRF increased
microvessel-like structure formation up to 700% and the addition
of high RCF PRF increased micro-vessel like structure formation
up to 250% after 14 days of cultivation. A statistically significant
higher percentage of angiogenic structures could be compara­
tively quantified in co-cultures treated with low RCF PRF com­
pared to co-cultures treated with high RCF PRF and untreated co-
cultures after 7 and 14 days. In addition, all experimental groups
showed a higher number of CD31 negative and DAPI-positive
cells (pOBs) assembling around CD31 positive microvessel-like
structures after 14 days of culture, whereas they were more uni­
formly distributed after 7 days of cultivation (Figure 4A).
Determination of growth factor content in co-cultured pOBs
and HDMECs treated with PRF
The protein concentrations of the growth factors TGF-β1, PDGF-
BB and VEGF were determined in supernatants of co-cultured
pOBs and HDMECs after 4, 7, 10 and 14 days (Figure 5). From
the 4th to the 14th day, increasing cumulative TGF-β1 concentra­
tions could be detected in all experimental groups. In general, co-
cultures treated with low RCF PRF revealed significantly higher
TGF-β1 levels compared to the other experimental groups after
a cultivation period of 4, 7 and 10 days, while they contained
similar amounts of TGF-β1 compared to high RCF PRF after 14
days of cultivation. When comparing co-cultures treated with
high RCF PRF and untreated co-cultures, co-cultures treated
with high RCF PRF revealed higher concentrations of TGF-β1
in cell culture supernatants at all time points evaluated. This was
statistically significant after a cultivation period of 10 days. After
10 days of cultivation, VEGF concentrations were only detectable
in supernatants of the control group and were assessed as statis­
tically significant higher compared to supernatants of co-cultures
treated with PRF. In turn, VEGF concentrations were statistically
significant higher in supernatants of co-cultures treated with PRF
compared to supernatants of untreated co-cultures after 14 days.
Statistically significant higher PDGF-BB concentrations could be
observed in supernatants of co-cultures treated with low RCF
PRF compared to the other experimental groups at all time points
evaluated. While no PDGF-BB concentrations were detectable in
supernatants of the other experimental groups, PDGF-BB content
was increasingly detectable in supernatants from the 4th to the
10th day in co-cultures treated with low RCF PRF and decreased
again on the 14th day.
proosteogenic and proangiogenic properties of PRF 7
Discussion
In order to improve wound healing and regeneration processes,
blood concentrates like platelet rich fibrin gained significant
attention in the field of dentistry and regenerative medicine
since PRF provides important immune cells and growth factors
with regenerative properties.5,10,17,18,24 Focus of the present study
was to further elucidate the potential of PRF paying particular
attention to the PRF mediated effect on osteogenic differentiation
and angiogenesis. Investigating the effect of PRF on human
primary osteoblasts as well as on a more complex tissue-like co-
culture consisting of osteoblasts and microvascular endothelial
cells, our study could demonstrate that PRF is able to support
and affect a variety of processes, including osteogenic prolifera­
tion, osteogenic differentiation as well as angiogenic structure
formation. Our results could assess a PRF mediated positive
effect on the proliferation of pOBs resulting in generally higher
cell numbers when pOBs were treated with PRF consistent other
studies, which showed a PRF-mediated higher number of viable
osteoblasts after 1 day of cultivation.25 Based on the present
results, application of low RCF PRF could be assessed as more
effective in increasing osteoblast proliferation than high RCF
PRF. This may be due to its higher concentrated cells such as
platelets and leukocytes and and their released growth factor
concentrations such as TGF-β and PDGF-BB that stimulate osteo­
blast to proliferate.26–28 These growth factors could be found to be
increased within supernatants of pOB treated with low RCF PRF.
Platelets not only evoke an proosteogenic potential through the
release of growth factors, but they have also been found to accel­
erate osteogenic proliferation through cellular components such as
microparticles and cell membranes. In addition, the fibrin matrix
of PRF may also attribute to an increase in proliferative capacity
under certain experimental circumstances, as previously outlined
in other studies.29,30 Besides the positive effect of PRF on osteo­
blast proliferation, the expression of osteogenic differentiation
factors like ALP, col-1, osteonectin, BMP-1 and BMP-2 mRNA
was found to be upregulated in response to PRF treatment after 7
days of culture. This phenomenon was particularly noticeable
when pOBs were treated with low RCF PRF instead of high
RCF PRF. This coincided with the release of platelet and leuko­
cyte derived growth factors TGF-β and PDGF-BB, which have
been shown to induce early osteogenic differentiation.26,31 Col-1
and ALP are characteristic markers for the early stage of osteo­
genic differentiation as they have already been found to be
expressed in preosteoblasts. ALP activity and the expression of
col-1 are increasingly upregulated according to the degree of
maturity of osteoblasts.32 Therefore, PRF may induce osteoblast
differentiation, which in turn results in the upregulated expression
of ALP and col-1. While our study revealed that ALP gene
expression was upregulated in response to PRF after 7 days of
cultivation, another study demonstrated a steady increase in ALP
activity expressed by PRF treated osteoblast over 28 days.33
Furthermore, due to the upregulated expression of col-1 mRNA,
a mediating role in bone matrix formation can be ascribed to PRF,
since col-1 is not only an important organic extracellular matrix
component of bone, but also provides a scaffold for the attach­
ment of apatite crystals.34 Another study also confirmed that
collagen-related proteins were upregulated by PRF treatment,
supporting our findings.35 In addition, growth factors present in
PRF such as TGF-β1 support this effect by positively stimulating
osteoblasts for collagen synthesis.36 BMP-1 and BMP-2 are
highly involved in bone formation and matrix mineralization by
affecting osteoblast differentiation, collagen maturation and the
accumulation of bone matrix components.26,37–39 Matrix miner­
alization is characteristic of the late stage of the osteoblastic
differentiation process and is influenced by factors such as
8 E. Dohle et al. Platelets, 2024; 35(1): 1–11

Figure 5. Evaluation of the release of the proangiogenic growth factors TGF-β1, VEGF and PDGF-BB from cell culture supernatants of co-
cultures consisting of pOB and HDMEC after 4, 7, 10 and 14 days. Statistical significance was calculated using ANOVA and was assessed by
*p < 0.05, **p < 0.01.

Osteonectin (ON), Osteopontin (OPN) and alkaline phosphatase
(ALP).32 The non-collagenous proteins ON and OPN are indica­
tive of mature osteoblasts and these proteins form a part of the
organic bone matrix where they facilitate matrix mineralization
by promoting the incorporation of hydroxyapatite crystals into the
bone matrix by specifically coupling organic collagen and inor­
ganic hydroxyapatite with their binding sites.40,41 In contrast to
ON, OPN expression tended to be downregulated, when PRF was
indirectly applied to pOBs. Since PRF apparently stimulates and
accelerates the osteogenic differentiation process, it is possible
that OPN expression might be beyond mRNA regulation at the
time measured and is already present as a finished protein. The
upregulation of osteogenic marker mRNA (ALP, OPN, col-1,
BMP-2) has been also demonstrated in a study that co-
cultivated neutrophils with osteoblasts and endothelial cells.42
Since PRF has been shown to contain leukocytes, osteogenic
differentiation could be also due to the action of these immune
cells. Previous studies have shown that the white blood cell
concentration is particularly higher in low RCF-PRF compared
to high RCF-PRF, supporting our finding that low RCF-PRF
DOI: https://doi.org/10.1080/09537104.2024.2316744
upregulates gene expression of osteogenic differentiation
factors.17,18 In addition, several other studies underlined the
PRF mediated effect on the processes of osteogenic differentiation
and mineralization, while the precise regulatory mechanisms of
PRF remain to be elucidated.33,35,43 In conclusion, it can be
supposed that PRF activates the osteogenic differentiation process
in pOBs towards more mature osteoblasts. Adequate tissue perfu­
sion is another essential factor for bone regeneration since vascu­
larization allows cell recruitment, growth factor delivery, removal
of waste products and the supply of nutrients and oxygens.44,45
For analyzation of the proangiogenic potential of PRF in a more
bone tissue like construct, our studies used an established co-
culture model consisting of pOBs and HDMECs.21 After
a cultivation period of 7 and 14 days, co-cultures were stained
by immunofluorescence for endothelial cell specific marker to
visualize microvessel-like structure formation. It could be
observed that PRF not only significantly stimulated the formation
of microvessel-like structures, but also reduced the time of this
formation. After 7 days of culture, co-cultures treated with low
RCF PRF already revealed the existence of microvessel-like
structures compared to the other experimental groups, where no
microvessel-like structure formation could be assessed. After
a 14-day incubation period, the amount of angiogenic structures
increased significantly in co-cultures treated with PRF compared
to the control co-cultures. Since there was a significant increase in
vascular structure formation in PRF-treated samples, it can be
concluded that vascularization was PRF-induced and not solely
the result of the co-culture itself. The PRF-mediated effect on
microvessel-like structure formation was more evident when low
RCF PRF instead of high RCF PRF was applied to the co-culture.
Other studies on the proangiogenic effect of PRF confirmed these
results.19,20,46 When PRF treated endothelial cells were co-
cultured with fibroblast, angiogenesis was markedly enhanced
and vessel formation was particularly more stimulated when co-
cultures were treated with low RCF PRF. Another study con­
firmed the potency of liquid PRF in promoting the formation of
microvessel-like structures in co-cultures composed of outgrowth
endothelial cells and pOBs. PRF contains several factors that
might have a positive impact on vessel formation like highly
concentrated cells such as platelets, leukocytes and their growth
factors all contributing to vessel formation.17,18 Using triple cul­
ture systems consisting of osteoblast, endothelial cells and neu­
trophils or macrophages, outgrowth endothelial cells and pOBs,
neutrophils and macrophages were found to positively stimulate
angiogenesis by releasing proangiogenic factors such as
VEGF.42,47,48 Other leukocyte and platelet derived growth factors
include TGF-β and PDGF that affect endothelial cell survival,
differentiation, proliferation and vessel stability, ultimately pro­
moting vascular structure formation.27,49–51 Since platelets and
leukocytes are more highly concentrated within low RCF PRF
compared to high RCF PRF the higher induction of newly estab­
lished microvessel-like structures may be due to their involve­
ment. Proangiogenic growth factors TGF-β1 and PDGF-BB were
found significantly higher in co-cultures treated with low RCF
PRF compared to co-cultures treated with high RCF PRF con­
firming this assumption. However, since the co-culture is
a dynamic process, VEGF may have been taken up by the
HDMECs and thus be consumed at the time of measurement as
revealed by another study explaining the low concentration of
VEGF within PRF/co-culture supernatants.19 During bone forma­
tion, osteoblasts release VEGF, which binds to receptors on the
surface of adjacent endothelial cells via paracrine signaling.52 In
turn, endothelial cells release osteogenic growth factors such as
BMP-2.53 This close communication of endothelial and osteo­
blasts becomes clear by immunoflourenscence staining as pOB
occupy a position adjacent to the sprouting endothelial cells
proosteogenic and proangiogenic properties of PRF 9
a.54,55 This underscores the importance of the interplay of osteo­
genic differentiation and angiogenesis in the context of bone
regeneration. PRF stimulates these processes by providing impor­
tant immune cells as wells as proangiogenic and proosteogenic
growth factors. Consequently, PRF, with its stimulating effect on
osteogenic differentiation and vascularization, combines a variety
of properties that are particularly relevant for improving regen­
eration processes. Since increased vascular density can also con­
tribute to tumorigenesis, it is important to consider this ulterior
motive in the clinical application of PRF.56 However, there is no
evidence in the literature that PRF promotes tumorigenesis or
tumor flareup in clinical applications. Additionally, this is cur­
rently the subject of further studies. To the best of our knowledge,
no other pathological side effects have been identified, rather PRF
has positive properties such as pain relief and scar
prevention.7,9,10 Since PRF treatment of monocultures and co-
cultures is characterized by a dynamic interplay of different cell
types and growth factors, it is difficult to exactly define the
precise molecular processes and influences exerted by PRF.
Although the present results provide insights into the potential
effects of PRF on the regeneration process, more detailed inves­
tigations with regard to PRF properties should be the subject of
future investigations. In conclusion, Platelet rich fibrin represents
a promising autologous tool for regenerative purposes as it pro­
vides important cells and growth factors associated with wound
healing. Based on the results, PRF seems to unites proosteogenic,
potentially osteoconductive and proangiogenic properties that
increased the processes of both osteogenic differentiation as
well as angiogenic structure formation. Consequently, PRF may
be utilized for applications in versatile fields of medicine in the
context of improving bone tissue regeneration.
Acknowledgments
The authors would like to thank Mrs. Verena Hoffmann for excellent
technical support.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
We gratefully acknowledge the funding from the Federal Ministry of
Education and Research (BMBF) and the joint project ‘FROST’
(13GW0447C) and of the Society of Blood Concentrates and
Biomaterials e.V. (SBCB e.V).
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