biomedicines

Article
Quality Assessment of Platelet-Rich Fibrin-Like
Matrix Prepared from Whole Blood Samples after
Extended Storage
Hideo Kawabata 1 , Kazushige Isobe 1 , Taisuke Watanabe 1 , Toshimitsu Okudera 1 ,
Masayuki Nakamura 1 , Masashi Suzuki 1 , Jietsu Ryu 1 , Yutaka Kitamura 1 , Hajime Okudera 1 ,
Kazuhiro Okuda 2 , Koh Nakata 3 and Tomoyuki Kawase 4, * ID
1 Tokyo Plastic Dental Society, Kita-ku, Tokyo 1140002, Japan; hidei@eos.ocn.ne.jp (H.K.);
kaz-iso@tc4.so-net.ne.jp (K.I.); watatai@mui.biglobe.ne.jp (T.W.); toshiokuderaphd@gmail.com (T.O.);
maoh4618@me.com (M.N.); g-yanagidouri-dental@crux.ocn.ne.jp (M.S.); ryu@cap.ocn.ne.jp (J.R.);
shinshu-osic@mbn.nifty.com (Y.K.); okudera@carrot.ocn.ne.jp (H.O.)
2 Division of Periodontology, Institute of Medicine and Dentistry, Niigata University, Niigata 9518514, Japan;
okuda@dent.niigata-u.ac.jp
3 Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, Niigata 9518520,
Japan; radical@med.niigata-u.ac.jp
4 Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University,
Niigata 9518514, Japan
* Correspondence: kawase@dent.niigata-u.ac.jp; Tel.: +81-252-627-559

Received: 20 August 2017; Accepted: 14 September 2017; Published: 18 September 2017

Abstract: The platelet-rich fibrin–like matrix (PRFM) is usually prepared onsite and immediately used
for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without
quality deterioration, we developed a method for preparation of PRFMs from short-term-stored
whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we
extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting
PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient
temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes
and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth
Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry,
and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no
significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs
were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a
time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the
coagulation activity may diminish, we propose that PRFMs can be prepared—without evident loss of
quality—from WB samples stored for up to 7 days by our previously developed method.

Keywords: platelets; platelet-rich fibrin; platelet-derived growth factor; fibrin fiber; storage

1. Introduction
Among the various types of platelet concentrates, the platelet-rich fibrin-like matrix (PRFM) has
been increasingly used as the most convenient biomaterial for regenerative therapy in dentistry [1].
Moreover, this popularity is supported by its multiple functions as both a matrix and scaffold and its
higher capacity for tissue regeneration than platelet-rich plasma (PRP) [2,3]. When compared with
other platelet concentrate subtypes, PRFM is usually expected to be prepared onsite as per patients’
needs, and immediately used for regenerative therapy. In practice, however, due to a patient’s physical

Biomedicines 2017, 5, 57; doi:10.3390/biomedicines5030057 www.mdpi.com/journal/biomedicines

Biomedicines 2017, 5, 57 2 of 11

condition or a doctor’s technical capabilities, PRP is extensively prepared on the day or just before a
surgical procedure.
In Japan, new regulations for regenerative medicine established in 2014 require all physicians and
dentists administering a regenerative therapy that involves a platelet concentrate to record and report
the preparation procedures and quality assessment data for PRFM preparations [4]. As a time-saving
measure, some physicians or dentists, mainly in private practice, outsource the PRFM preparation
process. Therefore, there is a need to develop an off-site PRFM preparation process.
Because anticoagulants, such as citrate and acid-citrate-dextrose (ACD), are added to whole
blood (WB) during collection, PRP can be prepared from stored blood and delivered the next day.
Even though some physicians or dentists intend to outsource PRFM preparation, due to a lack of
anticoagulants, PRFMs cannot be prepared off-site on the next day. Accordingly, another option is
to preserve their home-made PRFMs under appropriate conditions. However, there is no reliable
scientific evidence to support the safety and effectiveness of a preserved PRFM.
To circumvent this problem, in our previous study [5], we developed a technique for preparation
of PRFMs from WB samples stored short-term, and we validated their quality for use as a biomaterial
for regenerative therapy. In this previous study, however, we examined WB samples stored only for
up to 2 days. It is still unclear how long WB samples can be stored for PRFM preparation without
significant quality loss. In blood transfusion, platelet products can be stored for a maximum of 4–7 days,
depending on national guidelines and the type of product [6]. Therefore, it can be predicted that
platelets may not be useful for medical purposes after this expiry period. In this study, to evaluate
biological implications of the officially recommended period of storage for our purposes, we applied
our previously developed technique to WB samples stored for relatively long periods (≥5 days) and
assessed the quality of the resulting PRFMs.
To help readers correctly understand the identity of the fibrin matrix preparations used in this
study, we should emphasize the differences between our PRFM and Choukroun’s PRF: although
in a broad sense and judging by visual inspection, our PRFM is almost identical to Choukroun’s
original PRF prepared from freshly collected WB samples without anticoagulants, our PRFM may be
distinguished from original PRF by the use of both an anticoagulant and CaCl2 and the protocol for
concentrated growth factors (CGF) preparation in a narrow sense.

2. Experimental Section

2.1. Blood Collection, Preservation, and Platelet-Rich Fibrin–Like Matrix (PRFM) Preparation
The study design and consent forms for all procedures involving human participants were
approved by the ethics committee for human subjects at Niigata University School of Medicine in
accordance with the Helsinki Declaration of 1975 (revised in October 2008).
Blood samples (approximately 9.0 mL per tube) were collected from six nonsmoking healthy male
volunteers (age 32–68 years) using 21-gauge needles equipped with a conventional vacuum plain glass
tube (Plain BD Vacutainer Tube; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for immediate
PRFM preparation or with a vacuum plain plastic tube (Neotube; NIPRO, Osaka, Japan) for stored WB
samples as previously described [7–9].
For preparing a control PRFM by the conventional method, fresh WB samples were collected
into glass tubes in the absence of ACD-A (Terumo, Tokyo, Japan) and were immediately centrifuged
by means of a Medifuge centrifugation system (Silfradent S.R.L., Santa Sofia, Italy). This centrifuge
was designed to prepare CGF (which may be considered a member of the PRF family) and employs a
program that automatically changes the centrifugal speed as follows: 30”, acceleration; 2’, 2700 rpm
(600× g); 4’, 2400 rpm (500× g); 3’, 3000 rpm (800× g); and 36”, deceleration and stop [10].
For delayed preparation of PRFM, WB samples were collected into plastic tubes in the presence of
ACD-A and stored for up to 7 days at ambient temperatures (20–24 ◦ C) with gentle agitation using a
tube rotary mixer (NRC-20R; Nissin, Tokyo, Japan). At various time points, the stored WB samples were
Biomedicines 2017, 5, 57 3 of 11

transferred into glass tubes, warmed at 37 ◦ C, intermittently mixed with 200 µL (20 µL × 10 times) of
a 10% CaCl2 solution and centrifuged on the Medifuge centrifugation system. After elimination of the
red blood cell (RBC) fractions by forceps, the resulting PRFM samples were immediately compressed
with a stainless-steel PRFM compression device (PRF stamper® ; JMR Corp. Ltd., Niigata, Japan) [11]
and washed thrice with PBS for scanning electron microscopy (SEM) or stored without washing at
−80 ◦ C until determination of PDGF-BB levels.

2.2. Measurement of Glucose and Ca2+ Levels and pH

Prior to Ca2+ addition, the stored WB samples were quickly centrifuged at 415× g for 3 min
to obtain the plasma fraction, which was used to determine total free Ca2+ levels by means of a
commercial kit based on the MXB method (Calcium E-test Wako; Wako Pure Chemicals, Osaka, Japan)
as described elsewhere [5].
For PRFM preparation, the supernatant serum fractions obtained after centrifugation were
subjected to analysis of Ca2+ levels as described above and to quantification of glucose with a
commercial kit based on the GOD method (Glucose CII Test Wako; Wako Pure Chemicals) [5].
The serum fractions were also subjected to measurement of pH with pH indicators (MColorHast; EMD
Millipore Corp., Billerica, MA, USA) [5].

2.3. Quantification of a Growth Factor by an Enzyme-Linked Immunosorbent Assay (ELISA)

PDGF-BB levels were measured in the PRFM extracts using the Human PDGF-BB Quantikine
ELISA Kit (R&D Systems, Inc., Minneapolis, MN, USA) as previously described [8,11,12]. In brief,
individual PRFM samples were minced and homogenized for 1 min with sample tube size disposable
homogenizers (BioMasher II; Nippi, Tokyo, Japan). After centrifugation, the resulting supernatants
were analyzed by an ELISA.

2.4. Determination of Blood Cell Counts

The total number of blood cells in WB samples and in fractionated liquid samples was determined
in the same types of sample tubes and an automated hematology analyzer (pocH-100iV Diff; Sysmex,
Kobe, Japan) [5,13]. RBCs, white blood cells (WBCs), and platelets were counted either immediately
after blood collection or after storage, but before centrifugation.

2.5. Flow-Cytometric (FCM) Analyses

The platelet fraction was isolated from WB samples by centrifugation (530× g, 10 min), washed
twice with PBS, and resuspended in PBS at a density of 1–2 × 108 /mL. The platelets were incubated
with 10 mM adenosine 5’-diphosphate (ADP; Wako Pure Chemical, Osaka, Japan) or 0.1% CaCl2
(Wako) for 15 min at ambient temperature. To stop the reaction, an equal volume of a commercial
fixative, ThromboFix (Beckman-Coulter, Brea, CA, USA) was added to each platelet suspension
(100 µL) and incubated for 30 min. Platelets were then washed twice with PBS and probed with both a
phycoerythrin (PE)-conjugated mouse monoclonal anti-CD41 antibody and a fluorescein isothiocyanate
(FITC)-conjugated mouse monoclonal CD62P antibody (1:20) (BioLegend, San Diego, CA, USA) for
45 min at ambient temperature. After two washes with PBS, platelets were analyzed on a flow
cytometer (Cell Lab Quanta SC; Beckman-Coulter Inc., Brea, CA, USA) as previously described [14].
For isotype controls, mouse IgG1 (BioLegend) was employed.

2.6. Scanning Electron Microscopy

To examine the microstructure of fibrin fiber networks, PRFM samples were compressed, washed
thrice with PBS, and cut into small pieces. Then, the PRFM pieces were fixed with 2.5% glutaraldehyde,
dehydrated with a series of ethanol and t-butanol washes, freeze-dried, and finally examined by SEM
(TM-1000, Hitachi, Tokyo, Japan) with accelerating voltage 15 kV, as previously described [5,15].
Biomedicines 2017, 5, 57 4 of 11

2.7. Evaluation of Platelet Surface Antigen Expression by an Immunofluorescence Assay

Platelet concentrates were prepared from stored WB samples, rinsed, and resuspended in PBS in
sampleBiomedicines
tubes. Platelets
2017, 5, 57 were then treated with CaCl2 at a final concentration of 0.1% and incubated 4 of 11 for
15 min at ambient temperature. ADP (10 mM) served as a positive control [16]. After completion of the
2.7. Evaluation of Platelet Surface Antigen Expression by an Immunofluorescence Assay
required incubation time, the reaction was stopped by addition of ThromboFix (Beckman Coulter Inc.,
PlateletThe
Brea, CA, USA). concentrates
plateletswere
wereprepared
washed from stored
twice andWB samples, rinsed,
incubated and resuspended
with anti-human CD41 in PBS
or CD62P
in sample tubes. Platelets were then treated with CaCl2 at a final concentration of 0.1% and incubated
monoclonal antibodies (1:20; BioLegend, San Diego, CA, USA) (primary antibodies) for 40 min at
for 15 min at ambient temperature. ADP (10 mM) served as a positive control [16]. After completion
ambient temperature. Next, the platelets were again washed twice with PBS and were probed with a
of the required incubation time, the reaction was stopped by addition of ThromboFix (Beckman
secondary antibody,
Coulter a goat
Inc., Brea, CA, anti-mouse IgG H&L
USA). The platelets wereantibody (an Alexa
washed twice Flour® with
and incubated 555 conjugate;
anti-human 1:50;
CD41Abcam,
Cambridge,
or CD62PMA, USA), for
monoclonal 30 min (1:20;
antibodies at ambient
BioLegend,temperature. Finally,
San Diego, CA, after subsequent
USA) (primary antibodies)PBS
for 40washes,
the platelets
min at were
ambient mounted withNext,
temperature. an antifade mounting
the platelets medium
were again with PBS®and
(Vectashield
washed twice ; Vector Laboratories,
were probed
with a CA,
Burlingame, secondary
USA),antibody,
and CD41 a goat
andanti-mouse IgG H&L antibody
CD62P expression (an Alexa
levels were Flour®under
examined 555 conjugate;
a fluorescence
1:50; Abcam, Cambridge, MA, USA), for 30 min at ambient temperature. Finally, after subsequent
microscope equipped with a cooled CCD camera (Nikon, Tokyo, Japan).
PBS washes, the platelets were mounted with an antifade mounting medium (Vectashield ; Vector ®

Laboratories, Burlingame, CA, USA), and CD41 and CD62P expression levels were examined under
2.8. Statistical Analysis
a fluorescence microscope equipped with a cooled CCD camera (Nikon, Tokyo, Japan).
The results are reported as mean ± standard deviation (SD). For multigroup comparisons,
2.8.analyses
statistical Statistical were
Analysis
performed by one-way analysis of variance (ANOVA) (SigmaPlot 12.5; Systat
Software, Inc.,The San Jose,
results areCA, USA)aswith
reported meanBonferroni’s post hoc(SD).
± standard deviation test.For
Differences with
multigroup p-values < 0.05
comparisons,
statistical analyses were performed
were considered statistically significant. by one-way analysis of variance (ANOVA) (SigmaPlot 12.5;
Systat Software, Inc., San Jose, CA, USA) with Bonferroni’s post hoc test. Differences with p-values
<0.05 were considered statistically significant.
3. Results
3. Results
3.1. Time-Dependent Changes in The Characteristics of Whole Blood Samples
WB3.1. Time-Dependent
samples Changes
were stored in The
with Characteristics
gentle agitationofatWhole Bloodtemperature
ambient Samples because they were collected
into plain plastic tubeswere
WB samples and stored
storedwith
for up to 7agitation
gentle days. During thistemperature
at ambient period, both the platelet
because they wereand RBC
counts collected
did not change
into plainsignificantly (Figure
plastic tubes and stored1a,b).
for upAdditionally, WBC
to 7 days. During thiscounts did not
period, both shift, but
the platelet andrelative
RBC counts
percentages of WBC did subtypes
not changeunderwent
significantlymarked
(Figure 1a and b). Additionally,
alterations WBC The
(Figure 1c,d). counts did not shift,of small
percentages
and medium-size components of WBCs, such as lymphocytes, increased, whereas and
but relative percentages of WBC subtypes underwent marked alterations (Figure 1c thosed).ofThe
the large
percentages of small and medium-size components of WBCs, such as lymphocytes, increased,
components, such as granulocytes, decreased.
whereas those of the large components, such as granulocytes, decreased.

1. (a–c)1.Stable
Figure Figure counts
(a–c) Stable of platelets,
counts red red
of platelets, blood cells
blood (RBCs),
cells and
(RBCs), white
and whiteblood
bloodcells
cells (WBCs) in stored
(WBCs) in
whole blood samples (n = 8); (d) A comparison of WBC components between fresh and
stored whole blood samples (n = 8); (d) A comparison of WBC components between fresh and 7-day- 7-day-stored
WB samples. The data were calculated from an average of 8 samples. W-SCR: WBC small cell ratio,
W-MCR: WBC middle cell ratio, W-LCR: WBC large cell ratio.
Biomedicines 2017, 5, 57 5 of 11

stored
Biomedicines WB5,samples.
2017, 57 The data were calculated from an average of 8 samples. W-SCR: WBC small cell 5 of 11
ratio, W-MCR: WBC middle cell ratio, W-LCR: WBC large cell ratio.

Platelets’
Platelets’responses
responsestotostimulants
stimulantswere
wereevaluated
evaluated bybycomparing
comparing thethe
expression
expression of CD62P
of CD62Pwith that
with
of CD41 [17]. After storage for 2 days, CD41 expression was similar among all the samples,
that of CD41 [17]. After storage for 2 days, CD41 expression was similar among all the samples, regardless
of the external
regardless stimuli
of the (0.1%
external CaCl2(0.1%
stimuli or 10CaCl
mM2ADPor 10for
mM 15ADP
min;for
Figure 2). In
15 min; contrast,
Figure 2). InCD62P expression
contrast, CD62P
levels were levels
expression upregulated by the CaCl
were upregulated by2 or
theADP
CaCl challenge.
2 or ADP The 7-day
challenge. Thestorage
7-day duration
storage did not
duration alter
did
the platelet activation responses. CD62P expression levels were likewise increased
not alter the platelet activation responses. CD62P expression levels were likewise increased by by treatment with
similar concentrations
treatment with similarof CaCl2 and ADP.
concentrations of CaCl2 and ADP.

Figure 2. Immunofluorescent staining of CD41 and CD62P expressed in platelets isolated from 2-day-
Figure 2. Immunofluorescent staining of CD41 and CD62P expressed in platelets isolated from 2-day-
or 7-day-stored WB samples. (a,d) Control resting platelets; (b,e) platelets stimulated by 0.1% CaCl2
or 7-day-stored WB samples. (a,d) Control resting platelets; (b,e) platelets stimulated by 0.1% CaCl2
for 15 min; and (c,f) platelets stimulated by 10 mM ADP for 15 min. The platelets were derived from
for 15 min; and (c,f) platelets stimulated by 10 mM ADP for 15 min. The platelets were derived from
the same donor and were distributed with almost the same density in all the dishes (views).
the same donor and were distributed with almost the same density in all the dishes (views).
Similar observations were made during quantitative FCM analysis (Figure 3). In terms of
Similar
elevated observations
CD62P were
expression made
levels, during responsiveness
platelets’ quantitative FCM analysis
to ADP (Figure
or CaCl 3). Inatterms
2 stayed of elevated
constant levels
CD62P expression
with storage time. levels, platelets’ responsiveness to ADP or CaCl2 stayed at constant levels with
storage time.
In the liquid fraction of WB samples, Ca2+ levels remained similar throughout the storage period,
whereas glucose levels, mostly increased by ACD-A, decreased with storage time (Figure 4a,b).
Plasma pH stayed at 7.5 ~8.0 (Figure 4c).
 Biomedicines 2017, 5, 57 6 of 11

Biomedicines 5, 57 5, 57
2017,2017,
Biomedicines 66 of
of11
11

Figure 3. Flow-Cytometric (FCM) analysis of CD41- and CD62P-double-positive platelets in platelet

fractions that were prepared from fresh or stored WB samples and stimulated with 10 mM ADP or
0.1% CaCl2 for 15 min (n = 4). * p < 0.05 as compared with control platelets at the same time points. No
significant differences were observed in time-course changes.
Figure 3. Flow-Cytometric
Figure 3. Flow-Cytometric (FCM)(FCM) analysis of CD41-
analysis of CD41-and CD62P-double-positive
and CD62P-double-positiveplatelets
platelets in
in platelet
platelet
fractions
In the that were
liquidthat
fractions prepared
fraction from
of WB samples,
were prepared fresh
from fresh or stored
Ca orlevels
2+ WB samples
storedremained and
similar
WB samples stimulated
throughout
and stimulated with 10 mM
mM ADP
the10storage
with or
period,
ADP or
0.1% CaCl
whereas0.1%
glucose for
CaCl
2 15 min
2levels, (n =
mostly
for 15 min 4). * p <
increased
(n = 4). 0.05 as
* p < 0.05by compared with
ACD-A, decreased
as compared control platelets
with controlwith storage
platelets at the
time
at the same
same time
(Figure points.
4a and
time points. Nob).
No significant
Plasma significant
pH stayed differences
differences were
at 7.5 ~ 8.0 observed
observed
(Figure intime-course
4c). in time-coursechanges.
changes.

In the liquid fraction of WB samples, Ca2+ levels remained similar throughout the storage period,
whereas glucose levels, mostly increased by ACD-A, decreased with storage time (Figure 4a and b).
Plasma pH stayed at 7.5 ~ 8.0 (Figure 4c).

Figure 4. Stable Ca2+ (a) and glucose levels (b) and pH (c) of fresh and stored WB samples.
Because stored WB samples contained ACD-A as an anticoagulant, CaCl2 was added to the samples for
PRF clot formation. Ca2+ levels were determined before and after the addition of CaCl2 . Glucose levels
were determined in WB samples before the addition of CaCl2 . * p < 0.05 as compared with the individual
control levels on day 1 (n = 8).
Biomedicines 2017, 5, 57 7 of 11

Figure
Figure 4.4. Stable
Stable CaCa2+ (a)
2+
(a) and
and glucose
glucose levels
levels (b)
(b) and
and pH
pH (c)
(c) of of fresh
fresh and
and stored
stored WB
WB samples.
samples. Because
Because
stored
stored WB
WB samples
samples contained
contained ACD-A
ACD-A as as an
an anticoagulant,
anticoagulant, CaCl CaCl22 was
was added
added toto the
the samples
samples for
for PRF
PRF
clot
clotformation.
formation.Ca Ca2+levels
2+
levelswere
weredetermined
determinedbefore
beforeand
andafter
afterthe theaddition
additionofofCaCl
CaCl22..Glucose
Glucoselevels
levelswere
were
determined
Biomedicines 2017, 5,in
determined 57WB
in WB samples
samples before
before the
the addition
addition of CaCl22.. ** pp << 0.05
of CaCl 0.05 as
as compared
compared with with the
the individual
individual7 of 11
control levels on day 1 (n
control levels on day 1 (n = 8). = 8).

3.2. Time-Dependent
3.2.
3.2. Time-Dependent Changes
Time-Dependent Changes in
Changes in the
in the Quality
the Quality of
Quality of The
of The Resultant
The Resultant PRFM
Resultant PRFM Samples
PRFM Samples
Samples
Storage time
Storage
Storage timedid
time didnot
did not
notsubstantially
substantially
substantially affect thethe
affect
affect visual
the appearance,
visual
visual size, size,
appearance,
appearance, or serum
size, or retention
or serum
serum of PRFMs
retention
retention of
of
prepared
PRFMs from
PRFMs prepared stored
prepared from WB samples
from stored
stored WB (Figure
WB samples 5).
samples (FigureHowever,
(Figure 5). fibrin
5). However, fibers
However, fibrin formed
fibrin fibers in these
fibers formed
formed inPRFMs
in these became
these PRFMs
PRFMs
somewhat
became
became thinner thinner
somewhat
somewhat with time
thinner (Figure
with
with time 6).
time (Figure
(Figure 6).
6).

Visual appearance of
Figure 5. Visual
Figure of platelet-richfibrin–like
fibrin–like matrixs(PRFMs)
(PRFMs) preparedfromfrom WB samples
samples
Figure 5.
5. Visual appearance
appearance of platelet-rich
platelet-rich fibrin–like matrixs
matrixs (PRFMs) prepared
prepared from WB WB samples
stored
stored for the indicated periods. WB samples were simultaneously collected from the same donor.
stored for
for the
the indicated
indicated periods.
periods. WB
WB samples
samples were
were simultaneously
simultaneously collected
collected from
from the
the same
same donor.
donor.
Similar PRFM samples
Similar samples were obtained
obtained from three
three other experiments.
experiments.
SimilarPRFM
PRFM sampleswere were obtainedfrom
from threeother
other experiments.

Figure 6. Scanning electron microscopy (SEM) images of fibrin fibers formed in PRFMs prepared from
WB samples stored for the indicated periods. WB samples were simultaneously collected from the
same donor. Similar findings were obtained in three other experiments.
Biomedicines 2017, 5, 57 8 of 11

Figure 6. Scanning electron microscopy (SEM) images of fibrin fibers formed in PRFMs prepared from
WB samples stored for the indicated periods. WB samples were simultaneously collected from the
Biomedicines 2017, 5, 57 8 of 11
same donor. Similar findings were obtained in three other experiments.

PDGF-BB PDGF-BB
levelslevels
in thein extracts
the extracts
ofofthe
theresulting
resulting PRFM
PRFM samples significantly
samples decreased
significantly during the
decreased during the
initial 3 days but recovered to control levels thereafter (Figure
initial 3 days but recovered to control levels thereafter (Figure 7). 7).

Figure 7. Time-dependent changes in the concentration of PDGF-BB extracted from PRFM samples
Figure 7. Time-dependent
that were prepared fromchanges
stored WBinsamples
the concentration
and compressedoftoPDGF-BB
squeeze out extracted from* pPRFM
PRFM exudates. < 0.05 samples
that wereas compared
preparedwith fresh
from WB samples
stored as controls
WB samples and(n =compressed
8). to squeeze out PRFM exudates. * p <
0.05 as compared with fresh WB samples as controls (n = 8).
4. Discussion

4. Discussion
The biochemical mechanisms underlying different phases of platelet activation, including
adhesion, shape change, the granule release reaction, and aggregation, have been well delineated [18].
TheTobiochemical mechanisms
treat specific diseases, such as underlying
thrombocytopenia,different phases
functionally of platelet
complete plateletsactivation,
are required. including
adhesion, shape change,
However, in the PRFM the granule release reaction,
used for regenerative therapy,and aggregation,
platelets are only have been
required well delineated
to aggregate in [18].
2+
To treatresponse
specifictodiseases,
Ca and/or thrombin,
such to release growth factors,
as thrombocytopenia, and to support
functionally completeclot formation.
plateletsInare ourrequired.
previous study [7], we demonstrated that short-term storage does not influence the minimally required
However, in the PRFM used for regenerative therapy, platelets are only required to aggregate in
platelet functions or quality of the PRFM. The aim of this study was to investigate the possible expiry
response to Ca 2+ and/or thrombin, to release growth factors, and to support clot formation. In our
limit for the storage of PRFM. In general [19], the storage of platelets for clinical use is limited to a
previousmaximumstudy [7], we demonstrated
of 5 days. Consequently, we that did notshort-term storage
extend the storage does
period not
to >7 influence
days, the minimally
and we assessed
requiredtheplatelet
quality offunctions or quality
PRFMs prepared of the
from stored WBPRFM.
samples.The aim of this study was to investigate the
Both RBC and WBC counts tended
possible expiry limit for the storage of PRFM. In general to gradually, but [19],
not significantly,
the storagedecrease with storage
of platelets for clinical use
time, whereas platelet numbers did not. Regarding the time-dependent changes in Ca2+ , glucose,
is limited to a maximum of 5 days. Consequently, we did not extend the storage period to >7 days,
and PDGF-BB levels, as previously demonstrated [7], PDGF-BB levels in PRFMs prepared from WB
and we samples
assessed the quality of PRFMs prepared from stored WB samples.
stored for 1–3 days were significantly lower than those of fresh WB samples. Nonetheless, with
Both RBC and
increasing WBC
storage counts
time, tended
PDGF-BB levels torecovered
gradually, butof
to those not thesignificantly,
freshly prepared decrease
PRFM. Only with storage time,
glucose
whereaslevels platelet
changed numbers
with time;did not.
they Regarding
decreased the time-dependent
with increasing storage time, and changes
at 5 daysin andCa 2+ , glucose,
later, they and
were significantly lower than those of the ACD-treated WB samples
PDGF-BB levels, as previously demonstrated [7], PDGF-BB levels in PRFMs prepared from WB on day 1.
samples stored It is generally
for 1–3 days accepted
werethatsignificantly
adequate oxygen supply
lower than is needed
those of to increase
fresh WB platelet viability
samples. in
Nonetheless,
platelet concentrates because oxygen reduces their glucose consumption and lactate production [20,21].
with increasing storage time, PDGF-BB levels recovered to those of the freshly prepared PRFM. Only
It is known that even under the improved storage conditions, platelets gradually lose their function,
glucose alevels changed
phenomenon thatwith time;
is called thethey
storagedecreased with
lesion [22]. increasingit storage
Furthermore, was recentlytime, and at 5 days and
demonstrated
later, theythatwere
growth significantly
factors in PRP lower
degradethan in those of the
the course ACD-treated
of storage ◦
at 22 C WB[23]. samples
Therefore,on day 1.et al.
Bausset
It isrecommend
generallyinjecting
accepted PRPthat
within 3 h after oxygen
adequate preparation to avoid
supply the loss oftoefficacy
is needed increase[24].platelet
There is anviability in
opposite viewpoint, that PRP for tissue regeneration can be stored
platelet concentrates because oxygen reduces their glucose consumption and lactate production for at least 5 days [25].
In the present study, gas-impermeable plastic tubes were used for WB preservation, so that
[20,21]. It is known that even under the improved storage conditions, platelets gradually lose their
blood cell viability can be maintained mainly by glycolysis (as explained elsewhere [26,27]) of glucose
function, a phenomenon
provided by the ACD-A that solutionis [26,27]
calledafter the storage
depletion lesion [22].
of remaining oxygen.Furthermore,
In this case, evenitthoughwas recently
demonstrated that growth factors in PRP degrade in the course of storage at 22 °C [23]. Therefore,
Bausset et al. recommend injecting PRP within 3 h after preparation to avoid the loss of efficacy [24].
There is an opposite viewpoint, that PRP for tissue regeneration can be stored for at least 5 days [25].
In the present study, gas-impermeable plastic tubes were used for WB preservation, so that
Biomedicines 2017, 5, 57 9 of 11

platelets are not concentrated as highly as platelet concentrates for storage, it is possible that RBCs in
cooperation with platelets produce lactate and significantly decrease pH. On the other hand, plasma
pH of the stored WB samples remained constant at ~6.5 under our preservation conditions. As a result,
platelets could be preserved well, judging by the finding that the ability of the platelets—isolated
from 7-day-stored WB samples—to respond to Ca2+ and ADP challenges was mostly similar to that of
platelets obtained from the WB samples following short-term storage. A possible explanation for this
successful preservation may be suppression of cell metabolism by citrate-dependent Ca2+ chelation:
platelet activation is known to be prevented by citrate [28], whereas it is also possible that RBC activity
can be reduced through inhibition of Ca2+ -mediated cellular functions [29] by Ca2+ chelation.
Because WBC-depleted transfusion is necessary to avoid WBC-mediated adverse reactions,
particularly in allogeneic blood transfusions [30], the lifespan of WBCs in vitro has not been
clearly described in the literature: approximate lifespans of circulating RBCs, platelets, neutrophils,
eosinophils, and B cells are reported to be 120, 10, 1–5, and 2–5 days and 4–7 weeks, respectively [31].
Accordingly, granulocytes (large components of WBCs) may have the shortest survival period in vitro.
In contrast, lymphocytes, which constitute the small components of WBCs, and RBCs may survive
longer than can other blood cell types in vitro. Consistent with these standard lifespans, our present
findings indicate that the percentage of large WBCs decreased with time. Although WBC counts did
not significantly decrease within 7 days, it cannot be ruled out that the automatic hematology analyzer,
which uses particle size differences for calculations, may have detected and counted WBCs that were
reduced in size, probably by apoptosis.
The thickness of fibrin fibers of PRFM samples gradually decreased with time, probably due
to degradation of coagulation factors, a reduction in their enzymatic activity, or a decline of platelet
functions. Therefore, it can be hypothesized that PRFMs composed of thinner fibrin fibers may be
easily susceptible to degradation and may release growth factors faster than do fresh WB samples.
Nevertheless, fibrin fibers observed in fibrin clots that were prepared from fresh or frozen platelet-poor
plasma by the addition of thrombin were considerably thinner, and their cross-link density was
considerably higher relative to stored WB samples [7]. In our preliminary study, with a limited
number of WB samples, the degradation assay involving a trypsin and EDTA solution failed to detect
significant differences in PRFMs prepared from long-term–stored and fresh WB samples (data not
shown). We believe that the PRFM prepared from WB samples stored long-term can be an alternative
option for regenerative therapy in clinical settings.
Although blood transfusion studies have established standard protocols for the storage of WB
samples [32,33], the time-dependent reduction in WBC counts may result in weakened bactericidal
effects [34]. In addition, the possibility that autolysis of WBCs can trigger degradation of specific
proteins, which in turn can influence PRFM formation, cannot be ruled out. Consequently, to validate
the clinical use of such a PRFM, its safety and efficacy should be assessed further in experimental
models based on relatively large animals.

5. Conclusions
The PRFM is conventionally prepared onsite; however, it would be convenient if this material
were prepared several days later. We demonstrated that a clinically applicable PRFM can be prepared
from WB samples that are stored for up to 7 days by the addition of appropriate amounts of Ca2+ .
This method can make the treatment schedule more flexible and benefit both the patients and physicians
or dentists involved in regenerative therapy with the PRFM. Although the period of WB sample
storage may be further extended by improving several conditions, we would recommend using a fresh
autologous PRFM prepared onsite as the first choice and the PRFM prepared from stored autologous
WB samples as the second choice. To minimize the possible loss of efficacy and unidentified or
unpredictable risks, it would be better to utilize the stored autologous WB samples as soon as possible,
at least within a week in accordance with the national guidelines [6].
Biomedicines 2017, 5, 57 10 of 11

Author Contributions: Hideo Kawabata, Kazushige Isobe, Taisuke Watanabe and Tomoyuki Kawase
conceived and designed the study, performed the experiments, and wrote the manuscript. Kazushige Isobe,
Toshimitsu Okudera, Masayuki Nakamura, Masashi Suzuki, Jietsu Ryu, Yutaka Kitamura, and Kazuhiro Okuda
performed the experiments and data analysis. Hajime Okudera and Koh Nakata participated in manuscript
preparation. All authors read and approved the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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