International Journal of

Molecular Sciences

Article
Influence of Fractionation Methods on Physical and
Biological Properties of Injectable Platelet-Rich
Fibrin: An Exploratory Study
Prakan Thanasrisuebwong 1,2 , Rudee Surarit 3 , Sompop Bencharit 4,5 and
Nisarat Ruangsawasdi 6, *
1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Mahidol University, Bangkok 10400,
Thailand; [email protected]
2 Ph.D. program in Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand
3 Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand;
[email protected]
4 Department of General Dentistry, School of Dentistry, Virginia Commonwealth University, Richmond,
VA 23298, USA; [email protected]
5 Department of Biomedical Engineering, College of Engineering, Virginia Commonwealth University,
Richmond, VA 23298, USA
6 Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand
* Correspondence: [email protected]




Received: 27 February 2019; Accepted: 30 March 2019; Published: 3 April 2019


Abstract: Injectable platelet-rich fibrin (i-PRF) has been used as an autografting material to enhance
bone regeneration through intrinsic growth factors. However, fractionation protocols used to prepare
i-PRF can be varied and the effects of different fractionation protocols are not known. In this study,
we investigated the influence of different fractions of i-PRF on the physical and biological properties
derived from variations in i-PRF fractionation preparation. The i-PRF samples, obtained from
the blood samples of 10 donors, were used to harvest i-PRF and were fractioned into two types.
The yellow i-PRF fractionation was harvested from the upper yellow zone, while the red i-PRF
fractionation was collected from both the yellow and red zone of the buffy coat. The viscoelastic
property measurements, including the clot formation time, α-angle, and maximum clot firmness, were
performed by rotational thromboelastometry. The fibrin network was examined using a scanning
electron microscope. Furthermore, the concentration of growth factors released, including VEGF,
TGF-β1, and PDGF, were quantified using ELISA. A paired t-test with a 95% confidence interval
was used. All three viscoelastic properties were statistically significantly higher in the yellow i-PRF
compared to the red i-PRF. The scanning electron microscope reviewed more cellular components in
the red i-PRF compared to the yellow i-PRF. In addition, the fibrin network of the yellow i-PRF showed
a higher density than that in the red i-PRF. There was no statistically significant difference between
the concentration of VEGF and TGF-β1. However, at Day 7 and Day 14 PDGF concentrations were
statistically significantly higher in the red i-PRF compared to the yellow group. In conclusion, these
results showed that the red i-PRF provided better biological properties through the release of growth
factors. On the other hand, the yellow i-PRF had greater viscoelastic physical properties. Further
investigations into the appropriate i-PRF fractionation for certain surgical procedures are therefore
necessary to clarify the suitability for each fraction for different types of regenerative therapy.

Keywords: bone regeneration; fractionation; growth factors; i-PRF; PRF; ROTEM®

Int. J. Mol. Sci. 2019, 20, 1657; doi:10.3390/ijms20071657 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 1657 2 of 10

1. Introduction
Platelet-rich fibrin (PRF) derived from the centrifugation of whole blood has long been used
successfully in regenerative dentistry and medicine [1–5]. A centrifugation speed of 2700 rpm for
12 min in PRF with a glass or glass-coated tubes permits the use of PRF material without any
additional anticoagulants. The fibrin matrix scaffold network in PRF appears to be beneficial in tissue
regeneration [6]. PRF has shown to enhance regeneration of bone [1,7], cartilage [8], soft tissue [5,9–11],
and nerve fiber [12]. However, the application of solid PRF, resulting from a blood sample preparation
at 400 g relative centrifugal force (RCF) or 2700 round per minute (rpm) for 12 min, has its limitations.
For instance, it cannot be mixed with a particulate bone substitute. To mix with particulate bone,
the biomaterials in a liquid form are more suitable. Both injectable platelet-rich fibrin (i-PRF) and
PRF-predecessor platelet-rich plasma (PRP) are the liquid formula within the platelet concentrated
group that can be used efficiently for mixing with the particulate bone graft. PRP is traditionally
prepared from whole blood using a higher speed centrifugation compared to PRF [1–5]. Unfortunately,
PRP has some drawbacks because it requires either synthetic anticoagulants, such as citrate dextrose-A
(ACD-A) and citrate phosphate dextrose (CPD), or other activating agents, such as thrombin. Although
those additive agents allow for appropriate clinical working time and manipulation, they can inhibit
healing and regeneration due to the lack of biocompatibility [9–11,13–15].
The concept of low speed centrifugation is central to the preparation of the liquid form of i-PRF
as well as to create a material rich in leukocytes, platelets, and growth factors, including the vascular
endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), and platelet-derived
growth factor (PDGF), compared to the first generation platelet-derived PRP [9–11,16]. According
to the low speed centrifugation concept, i-PRF can be obtained by using a centrifugation speed at
60 g RCF or 700 rpm for 3 min [10,16,17]. Blood harvesting is similar to the PRF membrane method,
however, the non-coating plastic tube was recommended in order to prevent the formation of early
clotting in the tube. After centrifugation, whole blood is separated into three main parts based on
the buffy coat layer: A yellow upper part, a buffy coat middle part, and a red blood cell containing
lower part. A small syringe with an 18G hypodermic needle is used for collecting the i-PRF to be
used. The harvesting method of i-PRF after centrifugation has been described as harvesting only the
whole upper layer above the buffy coat [16,17], however, the amount of the upper layer harvested can
vary among individuals and the position of needle tips during harvesting can also be varied based
on different clinical practice. To the best of our knowledge, there is no study examining the different
properties of yellow and red i-PRF.
The aim of this study was to investigate the influence of different separation techniques of i-PRF on
its mechanical and biological properties. We further hypothesize that fractionation of the centrifuged
plasma for i-PRF is central to the i-PRF’s physical properties and biological components. We proposed
to examine two fractionation protocols producing yellow i-PRF and red i-PRF based on the fraction
of centrifuged plasma above and within the buffy coat, respectively. We expected that the yellow
i-PRF would have less cellular components, a denser fibrin network, and therefore have better physical
properties than the red i-PRF. On the contrary, we expected that the red i-PRF would have more cellular
components and therefore have better biological properties reflecting the greater release of known
PRF-related growth factors, including VEGF, TGF-β1, and PDGF.

2. Results
Rotational thromboelastometry (ROTEM® , Tem International GmbH, Munich, Germany) [18]
was applied to examine the viscoelastic properties of red and yellow i-PRF. The means and standard
deviations for the clot formation time (CFT), α-angle, and maximum clot firmness (MCF) were 52.8 ±
13.14 s, 81.2 ± 1.81 degree, and 85.3 ± 3.56 mm, respectively, for the yellow i-PRF and 68.2 ± 12.31 s,
77.8 ± 2.82 degree, and 81.6 ± 4.50 mm, respectively, for the red i-PRF as shown in Figure 1. The results
showed statistically significant differences (paired t-test) between the two types of i-PRF in all three
properties: CFT, α-angle, and MCF, with p-value = 0.008, 0.004, and 0.001, respectively, as shown in
 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 11

α-angle was higher in the yellow i-PRF compared to the red i-PRF. The MCF was also higher in the
yellow i-PRF compared to the red i-PRF.
The examination of the i-PRF by SEM showed the fibrin network architecture and cellular
Int. J. Mol.components.
Sci. 2019, 20, 1657In the yellow i-PRF, a dense fibrin network with less cellular components than the red 3 of 10
i-PRF was observed (Figure 2). On the contrary, SEM showed that more cellular components, such as
leukocyte, platelets, and erythrocytes, were in the red i-PRF compared to the yellow i-PRF. In
Figure 1.addition,
The CFT thefornumerous
the yellow erythrocytes
i-PRF was
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW
were enmeshed
shorter thaninthe
thered
fibrin network.
i-PRF. The shapes
The α-angle wasof higher
these in the
3 of 11
yellow i-PRFerythrocytes
compared were to normal, but i-PRF.
the red the fibrin
The network
MCF appeared
was alsoathigher
a lowerindensity and was
the yellow less organized
i-PRF compared to the
compared
α-angle was to higher
the yellow i-PRF
in the (Figure
yellow i-PRF 2).compared to the red i-PRF. The MCF was also higher in the
red i-PRF.
yellow i-PRF compared to the red i-PRF.
The examination of the i-PRF by SEM showed the fibrin network architecture and cellular
components. In the yellow i-PRF, a dense fibrin network with less cellular components than the red
i-PRF was observed (Figure 2). On the contrary, SEM showed that more cellular components, such as
leukocyte, platelets, and erythrocytes, were in the red i-PRF compared to the yellow i-PRF. In
addition, the numerous erythrocytes were enmeshed in the fibrin network. The shapes of these
erythrocytes were normal, but the fibrin network appeared at a lower density and was less organized
compared to the yellow i-PRF (Figure 2).

Figure 1. Rotational thromboelastometry (ROTEM® ) analysis demonstrating (A) clot formation time
(CFT), Figure 1. Rotational
(B) α-angle, and (C)thromboelastometry (ROTEM
maximum clot firmness ®) analysis demonstrating (A) clot formation
(MCF) of the yellow injectable platelet-rich fibrin
time (CFT), (B) α-angle, and (C)
(i-PRF) and the red i-PRF. ** p < 0.01. maximum clot firmness (MCF) of the yellow injectable platelet-
rich fibrin (i-PRF) and the red i-PRF. ** p < 0.01.
The examination of the i-PRF by SEM showed the fibrin network architecture and cellular
components. In the yellow i-PRF, a dense fibrin network with less cellular components than the
red i-PRF was observed (Figure 2). On the contrary, SEM showed that more cellular components,
such as leukocyte, platelets, and erythrocytes, were in the red i-PRF compared to the yellow i-PRF.
In addition, the numerous
Figure 1. Rotational erythrocytes were(ROTEM
thromboelastometry enmeshed in thedemonstrating
®) analysis fibrin network. The
(A) clot shapes of these
formation
erythrocytes
timewere normal,
(CFT), but the
(B) α-angle, andfibrin network
(C) maximum clotappeared at a lower
firmness (MCF) of the density and was
yellow injectable less organized
platelet-
compared rich fibrin
to the (i-PRF)
yellow and the
i-PRF 2). ** p < 0.01.
red i-PRF.
(Figure

Figure 2. Scanning electron microscope images of i-PRF using different fraction methods. A different
position of the harvesting needle led to two types of i-PRF: The yellow and red i-PRF. (A) The yellow
i-PRF showed a dense and highly organized fibrin network. (B) On the other hand, the red i-PRF
demonstrated more cellular components of leukocyte, platelets, and erythrocytes than the yellow i-PRF.
Int. J. Mol. Sci. 2019, 20, 1657 4 of 10

The growth factor concentrations were quantified at 3 h, 24 h, 72 h (3 days), 168 h (7 days), and
336 h (14 days) after preparation. There was a distinct difference between the releasing patterns
for VEGF, TGF-β1, and PDGF (Figure 3). At 3 h, 24 h, and 3 days, the VEGF concentrations were
increased. Three hours after preparation, the VEGF concentration of both i-PRFs was at a similar level.
This level presented in the yellow i-PRF was similar at 24 h again while the red i-PRF dramatically
increased. The concentrations of VEGF peaked at Day 3 for both the yellow and red i-PRF. There was a
slightly higher concentration of VEGF in the red i-PRF compared to the yellow i-PRF but the difference
was not statistically significant (Figure 3A). Different results were detected at Day 7 and 14 after
clotting. At this time point, both i-PRFs showed a decreasing VEGF concentration. In conclusion, the
released accumulation of VEGF showed a higher concentration in the red i-PRF than the yellow i-PRF
(Figure
Int. J. Mol.3B).
Sci. 2018, 19, x FOR PEER REVIEW 5 of 11

Figure 3. Time point releases and accumulated releases of three growth factors: (A,B) vascular
Figure 3. Time
endothelial point releases
growth and accumulated
factor (VEGF), releases of three
(C,D) transforming growth
growth factors: (A,B)
factor-β1 vascular
(TGF-β1), and (E,F)
endothelial growth factor (VEGF), (C,D) transforming growth factor-β1 (TGF-β1), and (E) and (F)
platelet-derived growth factor (PDGF). * p < 0.05.
platelet-derived growth factor (PDGF). * p < 0.05.
The growth factor concentration for the human TGF-β1 showed a general trend at all time points.
The growth factor concentration for the human TGF-β1 showed a general trend at all time points.
The highest concentration was recorded at 3 h for the yellow and red i-PRF then decreased and was
The highest concentration was recorded at 3 h for the yellow and red i-PRF then decreased and was
stable at 24 h, 3 days, and 7 days. There was a dramatic decrease of the TGF-β1 concentration for both
i-PRFs at Day 14 (Figure 3C). Note that the released accumulation of TGF-β1 presented a similar trend
and value for the yellow and red i-PRFs (Figure 3D). Using a paired t-test, there was however no
statistically significant difference between the concentration of VEGF and TGF-β1 (Figure 3).
The release of PDGF was more constant throughout the experimental timeline. Analysis of the
Int. J. Mol. Sci. 2019, 20, 1657 5 of 10

stable at 24 h, 3 days, and 7 days. There was a dramatic decrease of the TGF-β1 concentration for
both i-PRFs at Day 14 (Figure 3C). Note that the released accumulation of TGF-β1 presented a similar
trend and value for the yellow and red i-PRFs (Figure 3D). Using a paired t-test, there was however no
statistically significant difference between the concentration of VEGF and TGF-β1 (Figure 3).
The release of PDGF was more constant throughout the experimental timeline. Analysis of the
PDGF release (Figure 3E) revealed that more PDGF was released from the red i-PRF at 7 and 14 days
(975.47 ± 371.24 µg/mL and 615.98 ± 443.59 µg/mL) compared with the yellow i-PRF (623.46 ±
263.30 µg/mL and 385.59 ± 237.53 µg/mL). These data were statistically significant for Day 7 (p = 0.02)
and Day 14 (p = 0.03). Cumulatively, PDGF continued to release at all time points and a stronger
releasing trend was observed for the red i-PRF over the yellow i-PRF (Figure 3F).
Analysis of the kinetics of the growth factors released showed that VEGF, TGF-β1, and PDGF
reached peak release at Day 3, 3 h, and Day 7, respectively. On top of that, TGF-β1 was almost
completely released within the first 7 days while VEGF and PDGF showed a longer releasing over
14 days.

3. Discussion
The main difference of i-PRF from solid PRF is the lower speed and time in centrifugation
for i-PRF [10,19]. The idea of a slower centrifugation is to allow some cellular and growth factor
components to stay in the final product [6,9–11,17,20–24]. The i-PRF can be used in combination with
particulate bone allograft or autograft materials [25]. This advantage of a liquid formula of i-PRF
allows a more efficient incorporation of the bone graft material and increases the signaling molecules
throughout the whole graft.
Little information is known about the physical properties of i-PRF, which are important either
when being used alone or in combination with grafting materials. This study is one of the first to
apply ROTEM® technology, which is commonly used in hematology research [18,26–31], to examine
i-PRF. We hypothesized that compared to the lower layer—red i-PRF, the upper layer—yellow i-PRF
would have superior physical properties because of its lack of cellular components. All physical
properties confirmed our hypothesis. Although the yellow i-PRF was statistically superior compared
to the red i-PRF in viscoelastic properties, the value of the CFT, α-angle, and MCF of both i-PRFs
were similar to the reference value or slightly better compared to the standard whole blood that was
measured using ROTEM® [32]. In addition, the value of the CFT, α-angle, and MCF showed only a
minor difference between both i-PRFs. The SEM analysis demonstrated a dense, organized, acellular
fibrin network as a reason for the superior physical properties in the yellow i-PRF compared to the
red i-PRF. This clinically suggests that if i-PRF is to be used mainly as filler material or enhancement
stability and handling of grafting material, the yellow and red i-PRF may achieve a similar outcome.
A previous SEM analysis of leukocyte-PRF showed multi-different plasma layers that constituted
of a fibrin-rich layer at the uppermost layer, followed by an enriched platelet layer, and the buffy coat
layer with numerous leukocytes before the base layer of erythrocytes [33]. The SEM in our analysis
demonstrated a dense, organized, acellular fibrin network as in the fibrin-rich layer at the plasma
top layer of leukocyte-PRF. This characteristic might be a reason for the superior physical properties
in the yellow i-PRF compared to the red i-PRF. Meanwhile, the red i-PRF showed a greater number
of cells and platelets attached to the fibrin network similar to a combination of all the middle layers
and the erythrocyte base layer of leukocyte-PRF together. This additional cell and platelet content in
the red i-PRF might lead to better biological properties as we could observe a greater release of the
growth factors.
Contrary to the physical properties, we hypothesized that the red i-PRF, which has more cellular
components, would have higher release of growth factors. While there was no statistically significant
difference in the releases of VEGF and TGF-β1, PDGF at later time points were significantly higher
in the red i-PRF compared to the yellow i-PRF. This may be a result of the remaining platelet and
cellular components shown in the SEM in the red i-PRF. Note also that the releasing level of all three
Int. J. Mol. Sci. 2019, 20, 1657 6 of 10

growth factors was similar to previously reported levels [10]. In addition, we found that the growth
factors continue to release even at Day 14. This result is similar to other PRF studies [9–11,17,20–24].
Self-cooperation of the fibrin network slowly forms the high fibrillar aggregation in PRF, which also
entraps proteins and growth factors to the binding domains of fibrin molecules. In contrast, PRP is
rapidly activated to gelation from a load of thrombin. Therefore, an unstable fibrin network of PRP is
formed, which results in lower growth factor retention and incompatible cell homing. PRP therefore
only releases growth factors in the early stage. Unlike the PRP, the kinetic release of growth factors
from i-PRF is longer, up to 14 days, because of its preserved valuable components, including platelets
and leukocytes. The growth factor release from this component can be sustained over a longer period
of time from the cellular and acellular component trapped in a naturally progressive polymerization
of the fibrin matrix [34]. On top of this, i-PRF potentially allows more growth factor attachment to
the heparin-binding domain, which is the high-affinity growth factor-binding site of the fibrinogen
that causes prolonged retention of the growth factor within fibrin [35]. This result suggests that when
i-PRF is used in combination with particulate bone grafts, the signaling molecules for bone tissue
engineering can be achieved. The red i-PRF may therefore have more benefits than the yellow i-PRF
because of the greater release of the growth factor shown. In such cases, clinicians should harvest the
red i-PRF just with the buffy coat.
This study demonstrated for the first time that minimal changes in the fractionation protocol
for i-PRF could alter the physical and biological properties of the final product. This in turn may
change the clinical outcomes/applications. These techniques need to be examined further through
cell culture analysis and human clinical studies. In any case clinicians should pay particular attention
to centrifugation and fractionation protocols. Deviations from the published protocols can result in
variations of physical properties and biological components as seen in this study and in others [17].
In addition, further research to develop a novel biomaterial product from i-PRF is an interesting topic,
where the freeze-dried or lyophilized technique may be used for sterilization [36,37].

4. Materials and Methods

4.1. Sample Collection and Preparation

The study protocol was approved by the Institutional Review Board of Human Ethic Committee
of the Faculty of Dentistry, Mahidol University (COA. No. MU-DT/PY-IRB 2017/061.221). Blood
samples were harvested from 10 healthy volunteers (age range 30–35, gender M/F = 2/8). The donors
had no history of using antiplatelet or anticoagulant medications. All donors gave written consent for
blood collection and for sample preparation and experiments (Figure 4).
Sample preparation was adopted from a previous published protocol, Miron et al. 2017 [10].
Immediately after the blood sample was collected, the sample was centrifuged at 60 g RCF for 3 min
using a Duo Centrifuge (Process for PRF, Nice, France) at room temperature; 60 g RCF is equivalent to
700 rpm for this device, which has a 110 mm radius. After centrifugation, two types of i-PRF samples
were harvested, yellow and red i-PRF. According to Wang et al. 2017 [24], about 1 mL of sample, either
the yellow or red i-PRF, was collected. The yellow i-PRF referred to the sample harvested only in
the upper liquid yellow zone above the buffy coat. The red i-PRF referred to the sample harvested
from the zone, red and yellow, with the buffy coat. The bevel edge of the harvesting needle was used
as a reference point (Figure 4). Subsequently, both types of i-PRF were used to observe the physical
properties, including viscoelasticity and morphology, and the biological properties, which is the release
of growth factors.
4.1. Sample Collection and Preparation
The study protocol was approved by the Institutional Review Board of Human Ethic Committee
of the Faculty of Dentistry, Mahidol University (COA. No. MU-DT/PY-IRB 2017/061.221). Blood
samples were harvested from 10 healthy volunteers (age range 30–35, gender M/F = 2/8). The donors
hadInt.
noJ. history
Mol. Sci. 2019, 20, 1657
of using antiplatelet or anticoagulant medications. All donors gave written consent 7 of 10
for blood collection and for sample preparation and experiments (Figure 4).

Figure
Figure 4. Study
4. Study workflow
workflow of three
of three experiments
experiments comparing
comparing theand
the red red yellow
and yellow
i-PRF i-PRF and their
and their
fractionation protocol used in this study.
fractionation protocol used in this study.
4.2. Viscoelastic Property Analysis
Sample preparation was adopted from a previous published protocol, Miron et al. 2017 [10].
The after
Immediately viscoelastic
the blood properties of the i-PRF
sample was collected, samples
the sample were analyzed
was centrifuged using
at 60 g RCF for 3rotational
min
thromboelastometry (ROTEM ® ROTEM®
using a Duo Centrifuge (Process for ,PRF,
TemNice,
International GmbH,
France) at room Munich, Germany)
temperature; 60 g RCF is[18].
equivalent
generates
to 700 rpm foroutput from transducing
this device, which has achanges
110 mmreflecting fromcentrifugation,
radius. After the viscoelastic twostrength
types ofofi-PRF
the blood
sample
samples while
were a constant
harvested, rotational
yellow and red force is applied.
i-PRF. AccordingThree parameters
to Wang were[24],
et al. 2017 analyzed
about 1and mLdigitally
of
recorded:
sample, eitherClot
theformation
yellow ortime red(CFT),
i-PRF,α-angle, and maximum
was collected. clot firmness
The yellow (MCF). to
i-PRF referred Thethe
CFT represents
sample
harvested only
the speed at in the upper
which a solidliquid yellow
clot forms, zoneisabove
which the buffy
primarily coat. The
influenced by theredplatelet
i-PRF function,
referred to the
fibrinogen,
sample
and harvested
coagulation from the zone,
factors. TheredCFTandmeasures
yellow, with the buffyfrom
a duration coat.the
Theclot
bevel edge of the
initiation harvesting
until it reaches the
needle
20 mmwas amplitude.
used as a reference
The α-anglepointis(Figure 4). Subsequently,
a measure to observe the both types of of
dynamics i-PRF
clot were used towhich
formation,
represents the acceleration of the fibrin network formation and the amount of cross-linking build up.
The MCF is a measure for stability of the clot following the polymerization process.

4.3. Scanning Electron Microscopy (SEM)

The qualitative analysis of the morphological changes of the yellow and red i-PRF was performed
using SEM (JEOL, Peabody, MA, USA). The SEM examination was used to observe blood elements
present in the fibrin extracellular matrix. Cellular components such as leukocytes, platelets, and
erythrocytes were examined and recorded. The density of the fibrin network was also analyzed.
The i-PRF samples immediately after completing their clot formation were fixed in 2% glutaraldehyde
in Dulbecco’s phosphate buffered saline (DPBS) buffer with a pH of 7.4 overnight. Next, samples were
dried in the desiccator, then sputtered with 20 nm gold. SEM photography was then performed at 5 to
10 kV using 100× to 2000× magnifications.

4.4. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA kits were used to quantify the released growth factors: VEGF, TGF-β1, and PDGF (R&D
systems Minnesota, United States). Approximately 9 mL of collected i-PRF from each protocol was
transferred into a 6-well plate. The plate was then placed in a 5% CO2 incubator at 37 ◦ C for 30 min to
allow for complete clotting formation. Afterwards, 5 mL of Minimum Essential Medium Eagle-Alpha
Modification (α-MEM) with nucleosides (Gibco, USA) was added to each sample. The samples
were further incubated at 37 ◦ C to allow for the release of growth factors during a 3 h to 14 days
Int. J. Mol. Sci. 2019, 20, 1657 8 of 10

period of study. At 3 h, 24 h, 72 h (3 days), 168 h (7 days), and 336 h (14 days), 5 mL of culture
media was collected, frozen at −20 ◦ C, and replaced with 5 mL of additional fresh culture media.
The concentrations of VEGF, TGF-β1, and PDGF were measured according to the manufacturer’s
instructions. Optical density was assessed using a microplate reader at 450 nm. The measurements
were performed in triplicates.

4.5. Statistical Analysis

Statistical assessment was performed using SPSS version 17.0. All data were presented as
the mean value ± standard deviation (SD). The data were tested for the distribution by using a
Kolmogorov-Smirnov test and Levene’s test to confirm the normality and equal variance assumptions
of the data. The data, assuming normal distribution, were then analyzed using paired t-tests to
compare the differences between two groups in each experiment. The level of significance used was
p < 0.05.

5. Conclusions
This study suggests that fractionation protocols in i-PRF preparation can alter the final product’s
physical and biological properties. Clinicians should pay attention to the applications of i-PRF in
combinational use with other grafting materials. For the handling and stability of grafting material
application, the yellow and red may provide the final i-PRF product with a similar outcome. For the
use of i-PRF in enhancing biological properties, the red i-PRF collected from the sample with the buffy
coat, which released a higher level of growth factors, in particular, PDGF, should be used.

Author Contributions: Author P.T. designed the study, performed experiments, as well as collected and analyzed
the data. Author R.S. designed the study and interpreted the data. Author S.B. helped with study designs, data
interpretation, and manuscript writing. Author N.R. oversaw the entire project and supervised Author P.T. in all
experiments. All authors contributed to the manuscript writing and approved the final draft.
Funding: The study was funded by Ph.D. Research Grant, Faculty of Dentistry Mahidol University and it was
part of the research project supported by Mahidol University (043/2562).
Acknowledgments: We thank Chareerut Phruksaniyom for technical assistance and Center of Research, Faculty
of Dentistry, Mahidol University for providing devices.
Conflicts of Interest: The authors declare no conflict of interest.

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