Hindawi Publishing Corporation

Stem Cells International

Volume 2016, Article ID 7414036, 11 pages
http://dx.doi.org/10.1155/2016/7414036

Research Article
Platelet-Rich Plasma Obtained with Different
Anticoagulants and Their Effect on Platelet Numbers and
Mesenchymal Stromal Cells Behavior In Vitro

Ronaldo José Farias Corrêa do Amaral,1,2 Nemias Pereira da Silva,1

Natália Ferreira Haddad,1 Luana Siqueira Lopes,1
Fábio Dias Ferreira,1 Ricardo Bastos Filho,3 Paola Alejandra Cappelletti,1
Wallace de Mello,1,4 Eric Cordeiro-Spinetti,2 and Alex Balduino1,2
1
Excellion Serviços Biomédicos, Amil/UnitedHealth Group, 25651-000 Petrópolis, RJ, Brazil
2
Laboratório de Biologia e Tecnologia Celular, Universidade Veiga de Almeida, 20270-150 Rio de Janeiro, RJ, Brazil
3
Universidade Federal Fluminense, 24033-900 Niterói, RJ, Brazil
4
Centro Universitário Celso Lisboa, 20950-091 Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Ronaldo José Farias Corrêa do Amaral; [email protected]

Received 8 January 2016; Revised 9 April 2016; Accepted 27 April 2016

Academic Editor: Jiabing Fan

Copyright © 2016 Ronaldo José Farias Corrêa do Amaral et al. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.

There are promising results in the use of platelet-rich plasma (PRP) for musculoskeletal tissue repair. However, the variability in the
methodology for its obtaining may cause different and opposing findings in the literature. Particularly, the choice of the anticoag-
ulant is the first definition to be made. In this work, blood was collected with sodium citrate (SC), ethylenediaminetetraacetic acid
(EDTA), or anticoagulant citrate dextrose (ACD) solution A, as anticoagulants, prior to PRP obtaining. Hematological analysis
and growth factors release quantification were performed, and the effects on mesenchymal stromal cell (MSC) culture, such as
cytotoxicity and cell proliferation (evaluated by MTT method) and gene expression, were evaluated. The use of EDTA resulted
in higher platelet yield in whole blood; however, it induced an increase in the mean platelet volume (MPV) following the blood
centrifugation steps for PRP obtaining. The use of SC and ACD resulted in higher induction of MSC proliferation. On the other
hand, PRP obtained in SC presented the higher platelet recovery after the blood first centrifugation step and a minimal change in
MSC gene expression. Therefore, we suggest the use of SC as the anticoagulant for PRP obtaining.

1. Introduction factor beta (TGF-𝛽) [17], vascular endothelial growth factor

(VEGF) [18], and platelet-derived growth factor (PDGF) [19],
Platelet-rich plasma (PRP) is a blood-derived product in which have already been demonstrated to play important
which platelets are concentrated at least five times in plasma roles in tissue repair. When platelets are concentrated and
above the baseline of that in the whole blood [1]. PRP is being activated, it is expected that the concentration of the factors
investigated as an autologous product to improve tissue repair released reaches three to five times of that found in the plasma
in different conditions and lesions, especially for muscu- [16].
loskeletal tissues, such as chondral lesions [2–4], tendinopa- The general methodology to obtain PRP involves the col-
thies [5–7], muscle strains [8, 9], and bone repair [10, 11]. lection of whole blood with anticoagulants, followed by one
Besides its clinical application, PRP may be an efficient sub- or two centrifugation steps. After a first low-speed centrifuga-
stitute to fetal bovine serum in cell culture [12–15]. Its thera- tion, erythrocyte-free platelet concentrated plasma is recov-
peutic potential is based mainly on the growth factors present ered and submitted to high-speed centrifugation. Platelet-
in platelet’s alpha granules [16], such as transforming growth poor plasma is then discarded and the remaining platelet
2 Stem Cells International

pellet is homogenized into what is regarded as PRP [16]. to achieve the expected concentration of 106 platelets/𝜇L.
Several aspects on this method are still under debate, such The platelets in PRP2 were activated by adding 1 M CaCl2
as number of centrifugations, presence of leukocytes, and use (final concentration of 20 mM) and incubated at 37∘ C for
and type of platelet activator and anticoagulants [20]. When 1 hour. After clot formation, tubes were maintained at 4∘ C
no anticoagulant is used, a blood clot will form, and serum during 16 hours to allow clot contraction. Finally, tubes were
can be obtained but without increase in platelet concentration centrifuged at 3000 g for 20 minutes and the supernatant was
[21]. In the case of PRP obtaining, coagulation is not intended collected, termed as platelet-rich plasma releasate (PRPr).
to occur prior to platelet concentration; hence, blood must be The PRPr was freezed at −80∘ C until thawing for experimen-
collected in the presence of anticoagulants. tal use.
For transfusion purposes, blood is usually collected in
bags containing citrate phosphate dextrose adenine (CPDA- 2.3. Hematological Analysis. Counting of platelets, red blood
1) solution, as anticoagulant [22, 23], from which a platelet cells, white blood cells, and analysis of mean platelet volume
concentrate is obtained by double centrifugation of the whole (MPV) were determined in whole blood, PRP1, PRP2, and
blood or apheresis. Platelets concentrates obtained by such PPP fractions. Those analyses were performed with a hema-
methodologies may also be used for promoting tissue repair tological analyzer (Mindray BC 2800, Perdizes, SP, Brazil).
[24]. On the other hand, recent PRP formulations for autol- Platelet recovery after the first centrifugation step, expressed
ogous applications are usually prepared in collection tubes as a percentage, was calculated by dividing the total number
containing citrate solutions, in the form of sodium citrate of platelets in PRP1 by the total number of platelets in whole
[25–29] or ACD-A [30–32]. This last, ACD, is present in the blood.
majority of available commercial kits for PRP production [33,
34]. In other cases, heparin [35, 36] or EDTA [37] can be used. 2.4. Quantification of Growth Factors. PRPr-derived TGF-𝛽1
For clinical investigations, EDTA is commonly used in hema- and VEGF were quantified using ELISA kits (Ref: KAC1688
tology tests, SC in coagulation tests, and ACD in plasma levels and Ref: KHG0111; Invitrogen, Thermo Fisher Scientific)
measurement of platelet-derived components [38]. Therefore, according to manufacturer’s instructions. Absorbance was
our goal was to analyze how the choice of anticoagulant for determined using a microplate reader (Multiskan GO,
blood collection would modulate PRP characteristics as well Thermo Fisher Scientific).
as its effects on mesenchymal stromal cell culture.
2.5. Bone Marrow-Derived Mesenchymal Stromal Cell (BM-
2. Materials and Methods MSC) Isolation and Culture. 120 mL of bone marrow was
obtained after donation with informed consent from two
2.1. Ethics Statement. All of the experimental procedures donors (a 41-year-old woman, whose cells were used for cell
were approved by the Ethics Research Committee of viability assays, and a 60-year-old man, whose cells were used
the Pró-Cardı́aco Hospital, Rio de Janeiro (CAAE: for gene expression analysis). Mononuclear cells were sepa-
14878813.4.0000.5533), and all donors signed an informed rated using the Sepax system (Biosafe, Eysins, Switzerland),
consent. according to manufacturer’s instructions, and plated at 4 ×
105 cells/cm2 in Minimum Essential Medium Eagle Alpha
2.2. PRP Obtaining. PRP was obtained as previously described, Modification (alpha MEM) (Cultilab, Campinas, SP, Brazil)
with minor modifications [39]. Peripheral blood was col- supplemented with 10% fetal bovine serum (FBS) (Gibco)
lected from nine volunteer donors (6 men and 3 women) in T 150 cm2 culture flasks (Corning Incorporated, Corning,
using blood collection tubes containing sodium citrate (SC) NY) and maintained in a 5% CO2 incubator at 37∘ C. After
(Vacutainer, Ref: 369714; BD Biosciences, San Jose, CA), 5 days, medium was changed and nonadherent cells were
ethylenediaminetetraacetic acid (EDTA) (Vacutainer, Ref: discharged. Medium was changed every two days. This was
367861; BD Biosciences), or anticoagulant citrate dextrose termed as “primary culture.” After approximately 10 days, 70–
(ACD) solution A (Vacutainer, Ref: 364606; BD Biosciences) 80% confluence, cells were detached from culture flasks using
solution. The blood collected in one ACD tube was main- 0.05% trypsin solution (Gibco, Thermo Fisher Scientific)
tained in the same tube or divided into three polypropylene and replated onto new culture flasks at a density of 8 ×
tubes containing no anticoagulant (Falcon, Ref: 352063; BD 103 cells/cm2 . After first trypsinization, culture was termed as
Biosciences), termed as ACD-2. at “passage #1.” Experiments were performed until “passage
Tubes were centrifuged at 300 g for 5 minutes (Megafuge #5.”
40, Thermo Fisher Scientific, Waltham, MA). Supernatant
containing plasma and platelets, termed as platelet-rich 2.6. Cell Viability Assay. The analysis of cell viability was
plasma 1 (PRP1), was collected from each tube and transferred performed by incorporation with thiazolyl blue tetrazolium
to new polypropylene tubes containing no anticoagulant. In bromide (MTT assay) (Sigma Aldrich, São Paulo, SP, Brazil).
the case of ACD and ACD-2, after platelet counting, PRP1 Cells (passage #3) were plated at a density of 5 × 103 cells/cm2
from the same donors was mixed for the next experiments. in duplicate in 48-well plates (Corning Incorporated) in alpha
Then, PRP1 was centrifuged at 700 g for 17 minutes. Super- MEM (Cultilab) supplemented with 10% FBS (Gibco), 1%
natant was collected, namely, platelet-poor plasma (PPP). Part PRPr, 2.5% PRPr, or 5% PRPr. Four different PRPr donors
of the PPP from each tube was used to resuspend the platelet were used. In another group, the four samples were pooled
pellet, forming the platelet-rich plasma 2 (PRP2), in order with equal proportions of each donor, namely, PRPr MIX.
Stem Cells International 3

After 8 days of culture, 0.5 mg/mL MTT was added. Medium tubes. ACD containing tube was bigger—taller and larger—
was removed after 4 hours of incubation, and 400 𝜇L/well of compared to EDTA and SC.
DMSO was added to dissolve the reduced formazan product. In order to verify if the lower platelet recovery was related
The volume in each of the 48 wells was split into two wells in to the tube format, PRP was obtained from blood samples
a 96-well plate (Corning Incorporated). Finally, the plate was anticoagulated in ACD using tubes of similar size compared
read in a microplate reader (Multiskan GO, Thermo Fisher to EDTA and SC (ACD-2). Platelet recovery improved
Scientific) at 570 nm. Cell culture medium was not changed (49.82%) but remained much lower than those recovered
during this experiment. when using EDTA (76.15%) and SC (81.21%). Values from
ACD-2 were statistically different from those obtained using
2.7. Gene Expression Evaluation. Cells (passage #5) were cul- SC (𝑝 < 0.05) but not when compared to EDTA (𝑝 > 0.05)
tured in alpha MEM (Cultilab) supplemented with 10% FBS (Figure 1(b)). If analyzed separately, it was possible to observe
(Gibco), 1% PRPr, 2.5% PRPr, or 5% PRPr. The PRPr was used that platelet recovery has increased in three of the five donors
as a pool of four different donors. After five days of culture, when using ACD-2 instead of ACD, especially in donor 2,
total RNA was extracted using TRIzol (Ambion, Thermo with an increase of 63.74%, while it has decreased in two
Fisher Scientific). RNA concentration was determined using donors, especially in donor 1, with a decrease of 25.48% after
a Nanodrop 2000 UV-Vis spectrophotometer (Thermo Fisher the distribution of blood into the smaller tubes (Figure 1(c)).
Scientific) and 2 𝜇g was reverse-transcripted into comple- In average, platelet concentration in PRP2 was 1,009 ± 57 ×
mentary DNA (cDNA) using SuperScript First-Strand Syn- 103 /𝜇L in EDTA samples, 582 ± 108 × 103 /𝜇L in SC samples,
thesis System for RT-PCR (Invitrogen, #11904-018) in a total 726 ± 200 × 103 /𝜇L in ACD samples, and 664 ± 170 × 103 /𝜇L in
reaction volume of 20 𝜇L, following manufacturer’s proto- ACD-2 samples. All values were statistically similar between
col. Oligonucleotides and probes for qPCR were purchased each other (𝑝 > 0.05), except between EDTA and SC (𝑝 <
from Applied Biosystems (TaqMan Gene Expression Assay, 0.05) (data not shown).
#4331182): HPRT1 (Hs02800695 m1), which was analyzed Although the mean platelet volume (MPV), which is
as the housekeeping gene, SOX9 (Hs00165814 m1), RUNX2 related to platelet size and indicates its degree of activa-
(Hs00231692 m1), PPARG (Hs01115513 m1), and POU5F1 tion, was similar when whole blood (WB) samples were
(Oct-4) (Hs0099634 9H). qPCR reactions were performed in anticoagulated in all three anticoagulants tested, it increased
an Applied Biosystems 7500 Standard Time PCR System in a progressively following the two centrifugation steps in EDTA
20 𝜇L reaction volume using TaqMan Universal Master Mix group in all donors (in average an increase of 11.60% after the
II, with UNG (Applied Biosystems, #4440038), according to first centrifugation step and an additional increase of 2.84%
manufacturer’s instructions. Analysis was performed using after the second centrifugation step, totaling 14.44% increase
the ΔΔCt method [40]. compared to whole blood). This was not observed when WB
was anticoagulated in SC and ACD (Figure 2).
2.8. Statistical Analysis. Data were analyzed using a two- 3.2. TGF-𝛽1 and VEGF Release from Platelet-Rich Plasma in
tailed paired 𝑡-test for the hematological analysis, where Different Anticoagulants. Up to this point, it was clear that
a group of the same donors were analyzed with different the anticoagulant has an impact on platelet recovery after
anticoagulants. In the case of growth factor quantification and blood centrifugation. However, we questioned if it would also
cell culture experiments, where pairing of samples did not change growth factors release from recovered platelets. For
necessarily occur, a two-tailed unpaired 𝑡-test was performed. that, we quantified TGF-𝛽1 and VEGF levels in an ELISA
Statistical significance was considered when 𝑝 < 0.05. assay. Growth factors concentrations were similar between
anticoagulant groups (𝑝 > 0.05) for both TGF-𝛽1 and VEGF.
3. Results TGF-𝛽1 concentration was 18,146.99 ± 2,370.33 pg/mL in
EDTA; 48,559.10 ± 12,839.86 pg/mL in SC; and 30,786.15 ±
3.1. Effect of Different Anticoagulants on Initial Platelet Count- 6,654.49 pg/mL in ACD (Figure 3(a)). VEGF concentration
ing and Recovery. Blood samples were collected from five was 278.88 ± 71.78 pg/mL in EDTA, 143.65 ± 71.63 pg/mL in
different donors in tubes containing EDTA, SC, or ACD, SC, and 362.70 ± 77.95 pg/mL in ACD (Figure 3(b)).
and platelets were counted in an automated system. Blood
samples collected with EDTA yielded higher numbers of 3.3. Bone Marrow-Derived Mesenchymal Stromal Cell Culture.
platelets, followed by SC and ACD (Figure 1(a)). In average, In order to show the effects of factors released from platelets
platelet counting in SC was 16.28% lower than that in EDTA, obtained using different anticoagulants on modulating cell
while that in ACD was 23.01% lower than in EDTA and 7.94% expansion in vitro, we used the MTT cell viability assay to
lower than in SC. However, platelet recovery, regarding the analyze BM-MSC proliferation in the presence of different
total number of platelets obtained after the first centrifugation concentrations of PRPr. FBS-supplemented culture medium
step, was higher in the presence of SC compared to EDTA and was used as reference (Figure 4). PRPrs were tested separately
ACD. The average of platelet recovery in EDTA and SC was and mixed (MIX). All concentrations of PRPr tested from
76.15% and 81.21%, respectively. Strikingly, platelet recovery all donors were able to stimulate cell proliferation in vitro.
in samples collected with ACD was 45.71%, almost half of As expected, the higher concentration tested (5%) stimulated
those when using EDTA or SC. All three anticoagulants the higher proliferative rate in vitro, regardless of the anti-
tested herein were purchased in commercially distributed coagulant used. However, for this concentration, in average,
4 Stem Cells International

350 100
∘ ∘
90 +
300
80
250 +
Platelets (×103 /𝜇L)

Platelet recovery (%)

70 ∗
∗
200 60
50
150
40
100 30
20
50
10
0 0
Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 EDTA SC ACD ACD-2

EDTA ACD SC
(a) (b)
80

70

60
Platelet recovery (%)

50

40

30

20

10

0
ACD ACD-2

Donor 1 Donor 4
Donor 2 Donor 5
Donor 3 Average
(c)

Figure 1: Platelet yield and recovery in blood collected with different anticoagulants. Blood was collected in EDTA, SC, and ACD in five
different donors and platelet concentration was quantified (a) as well as platelet recovery after the first centrifugation step (b). An individual
analysis between ACD and ACD-2 of platelet recovery was also performed (c). Data are expressed as bar (a), box (b), and dot (c) plots. Similar
symbols in (b) correspond to statistic similarity among groups (𝑝 > 0.05).

cell proliferation was lower in the presence of EDTA derived observing the maximum and minimum relative quantifica-
PRPr compared to SC and ACD (Figure 4). In addition, cells tion of gene expression, the SC group presented the smallest
maintained their fibroblast-like morphology regardless of the variation compared to the control group, by analyzing the
anticoagulant type (Figure 5). average relative expression of the four genes in the PRPr
groups compared to the FBS group. In average, SC relative
3.4. Modulation of Bone Marrow-Derived Mesenchymal Stro- gene expression was 24.73% different from the control group,
mal Cell Gene Expression by Platelet-Rich Plasma Culture. while EDTA was 46.79% and ACD was 29.74% different.
We also analyzed gene expression of cells expanded in vitro
(Figure 6). Only cells cultured in 5% PRPr were tested. Using 4. Discussion
10% FBS as reference, RUNX2 was slightly upregulated in
EDTA group and downregulated in SC group. PPAR𝛾2 was Platelet-rich plasma (PRP) is currently one of the main
slightly upregulated in EDTA group and downregulated in strategies to promote musculoskeletal tissues repair. There
SC and ACD groups. SOX9 was downregulated in all groups. are several reports in the literature evidencing its potential
Oct-4 was upregulated in EDTA and ACD groups and down- in clinical trials [2–11] as well as in vitro analysis [12–15]. As
regulated in SC group. Although, in general, the gene expres- a cost-effective source of autologous growth factors that can
sion was similar between the PRPr groups, especially when affect stem cells proliferation and differentiation, it is being
Stem Cells International 5

Donor 1 Donor 2
11.5 11.5

11 11

Mean platelet volume (fL)

Mean platelet volume (fL)

10.5 10.5

10 10

9.5 9.5

9 9

8.5 8.5

8 8
WB PRP1 PRP2 WB PRP1 PRP2

EDTA ACD SC EDTA ACD SC

Donor 3 Donor 4
11.5 11.5

11 11

Mean platelet volume (fL)

Mean platelet volume (fL)

10.5 10.5

10 10

9.5 9.5

9 9

8.5 8.5

8 8
WB PRP1 PRP2 WB PRP1 PRP2

EDTA ACD SC EDTA ACD SC

Donor 5 Average
11.5 11.5

11 ∗
11 ∗
Mean platelet volume (fL)
Mean platelet volume (fL)

10.5 10.5

10 10

9.5 9.5

9 9

8.5 8.5

8 8
WB PRP1 PRP2 WB PRP1 PRP2

EDTA ACD SC EDTA ACD SC

Figure 2: Mean platelet value quantification of samples containing different anticoagulants. Mean platelet value was quantified in five different
donors in whole blood (WB), PRP1, and PRP2, in tubes containing EDTA, SC, or ACD solution. The average values of the five different donors
are also represented in the figure. “∗” corresponds to statistical difference between EDTA and SC groups as well as EDTA and ACD groups
(𝑝 < 0.05).

increasingly investigated as a supplement, adjuvant, carrier, to verify PRP obtaining with three types of commercially
or scaffold for stem cells-based therapeutics [41–45]. How- available blood collection tubes containing EDTA, SC, or
ever, the lack of standardization between the methodology to ACD as anticoagulants.
obtain and use PRP among different groups may hamper the Platelet counting was higher in blood collected in tubes
development of this technology [20]. The use of anticoagulant containing EDTA, followed by SC and ACD. Indeed, it has
to collect blood is a major issue. The present work aimed been previously shown that platelet count in EDTA can be
6 Stem Cells International

70.000 500

450
60.000
400
50.000 350
TGF-𝛽1 (pg/mL)

VEGF (pg/mL)
300
40.000
250
30.000
200

20.000 150

100
10.000
50

0 0

EDTA ACD SC EDTA ACD SC

(a) (b)

Figure 3: Growth factor quantification in PRPr obtained in different anticoagulant. TGF-𝛽1 (a) and VEGF quantification (b). Data are
expressed as mean, and error bars correspond to standard error.

higher than in citrate anticoagulants [46]. Moreover, when use in certain countries [54]. ACD and citrate-theophylline-
EDTA is added to citrated samples, it can enhance platelet adenosine-dipyridamole (CTAD) are also more efficient in
count in whole blood [47]. Platelet recovery after the first cen- maintaining platelet morphology than heparin and SC. In
trifugation step was diminished in ACD tubes compared to that case, it has also been shown that PRP obtained with
EDTA and SC. Additionally, a higher concentration of PDGF- ACD and CTAD resulted in higher TGF𝛽-1 concentration
BB was found in PRP obtained with EDTA compared to ACD and induction of MSC proliferation [55]. In another previous
[48]. In our case, we tried to enhance platelet recovery in ACD study, EDTA, SC, and ACD were compared as maintainers
tubes by dividing its content into smaller tubes (12 × 75 mm × of platelet responsiveness to aggregation inducers. ACD was
5 mL) with no additional anticoagulant. Although no statis- the most capable to maintain intraplatelet signal transduction
tical difference has been detected between those two ACD mechanisms during PRP formulation [56]. The same group,
forms, the splitting of blood in the smaller tubes resulted in
lately, showed that ACD was also capable of maintaining
a similar platelet recovery compared to EDTA group. Since
platelet functions for periods of time superior to SC [57]. In
ACD tube is bigger (16 × 100 mm × 8.5 mL) than EDTA (13 ×
our case, we could not find any difference in the capability of
75 mm × 4.0 mL) and SC tubes (13 × 75 mm × 4.5 mL), it is
possible that the lower platelet recovery is due not only to platelet activation and clot formation in PRP2 obtained with
the type of anticoagulant itself but also to the tube format. the three different anticoagulants.
Particularly, the tube format may have superior influence Our next step was to evaluate the effect of PRP obtaining
on whole blood/plasma than serum centrifugation, in view in cell culture. For that, we normalized the platelet concen-
of its higher viscosity [49]. For the following experiments, tration to ideally 1000 × 103 /𝜇L in all groups, in a way that
we decided to mix ACD and ACD-2 PRP1 from the same the results would not correlate to platelet concentration but
donors, since it is comprised of the same type of anticoagulant to the type of the anticoagulant. This platelet concentration in
and since the following centrifugation to prepare PRP2 was PRP is being pointed to as a therapeutic concentration for in
performed in the same type of tube for all groups analyzed. vivo purposes [1]. In bone, for example, lower concentrations
In order to verify if the centrifugation steps influenced are unsatisfactory and higher concentrations are inhibitory
platelets morphology, we quantified the mean platelet volume to promote tissue repair [11]. The only statistical difference
(MPV) in whole blood, PRP1, and PRP2 obtained with dif- observed in platelet concentration in PRP2 was between
ferent anticoagulants. The MPV in the EDTA group, but not EDTA and SC group. The lower value in SC group may be
in SC and ACD groups, has increased after the centrifugation attributed to a difficulty to resuspend platelet pellet. Platelet
steps, which may be an indicator of platelet activation [50, 51]. concentrate bags prepared with citrate samples, in form
Indeed, a higher MPV is expected in whole blood collected of ACD, contain more aggregates than bags prepared with
in EDTA compared to citrated samples [46]. In addition, EDTA [52]. Moreover, the effects of PRPr on cell culture were
EDTA may change platelet morphology from a discoid to always compared to 10% FBS medium supplementation. In
an irregular spherical shape [52]. Indeed, the use of EDTA spite of being a xenogeneic serum with ethical and scientific
faces ethical issues, such as being pointed to as a persistent issues [58], FBS is still widely used in cell culture [59] and
pollutant in natural environments [53], and impediments of MSC in vitro expansion [60].
Stem Cells International 7

0.4 0.4

0.35 0.35
∗ ∗
0.3 0.3
Absorbance (570 nm)

Absorbance (570 nm)

∗
0.25 0.25

0.2 0.2

0.15 0.15

0.1 0.1

0.05 0.05

0 0
Donor 1 Donor 2 Donor 3 Donor 4 MIX Donor 1 Donor 2 Donor 3 Donor 4 MIX

1% PRPr 5% PRPr 1% PRPr 5% PRPr

2.5% PRPr 10% FBS 2.5% PRPr 10% FBS
(a) (b)
0.4

0.35
∗
0.3
Absorbance (570 nm)

0.25

0.2

0.15

0.1

0.05

0
Donor 1 Donor 2 Donor 3 Donor 4 MIX

1% PRPr 5% PRPr
2.5% PRPr 10% FBS
(c)

Figure 4: BM-MSC viability in PRPr obtained with different anticoagulant. Absorbance at 570nm was measured after MTT viability assay
of cells cultivated in different PRPr concentrations, obtained with EDTA (a), SC (b), and ACD (c), as well as 10% FBS (control). Data are
expressed as mean, and error bars correspond to standard error. “∗” corresponds to statistical similarity with 10% FBS (𝑝 > 0.05).

In our study, we could not find differences in TGF𝛽- on growth factors and other molecules content among each
1 and VEGF concentration among the anticoagulants. It platelet granule, an evident pattern emerged: the concentra-
could be possibly due to the similar platelet concentration tion of 5% PRPr in cell culture medium was sufficient to
between groups. In average, TGF-𝛽1 concentration varied induce cell proliferation in a similar level to 10% FBS. Addi-
from 18.15 ng/mL in EDTA to 48.56 ng/mL in SC. Other liter- tionally, SC and ACD-derived PRPr presented greater effects
ature reports present concentrations superior to our finding: over cell proliferation compared to EDTA group. Cell mor-
120 ng/mL [61], 169 ng/mL [62], and 89 ng/mL in a PRP with phology was not changed among groups. BM-MSC main-
platelet concentration 4.69 times superior to whole blood tained their fibroblastic morphology regardless of the antico-
baseline but 20 ng/mL in a PRP with platelet concentration agulant. Although there are still controversies in the literature
1.99 times superior to whole blood baseline [63]. VEGF con- regarding PRP effects on BM-MSC differentiation, there is a
centration ranged from 143.65 pg/mL in SC to 362.70 pg/mL consensus on its effect as an inducer of proliferation [66].
in ACD. Other reports present concentrations varying from As a final analysis of PRPr effects on BM-MSC, we
50 pg/mL [64] to 155 ng/mL [61]. Nevertheless, our results observed slight modulations in the expression of the master
were superior to some commercially available kits [65]. genes for the osteogenic (RUNX2), adipogenic (PPAR𝛾2),
As expected, PRPr induced cell proliferation. Although and chondrogenic (SOX9) lineages [67], as well as Oct-4, a
there were variations comparing the influence on cell prolifer- gene related to maintenance of stemness [68, 69]. Particu-
ation between donors, possibly due to an inherent difference larly, SOX9 expression was downregulated. Indeed, there is a
8 Stem Cells International

(a) (b)

(c) (d)

Figure 5: Photomicrography of BM-MSC. Cells cultivated for eight days in medium supplemented with 10% FBS (a) or a pool from four
donors of 5% PRPr obtained from collection tubes containing EDTA (b), SC (c), or ACD (d). Phase contrast, 200x magnification, and scale
bars: 50 𝜇m.

2.5 1.6
PPAR𝛾2 mRNA relative expression
RUNX2 mRNA relative expression

1.4
2
1.2

1.5 1
0.8
1 0.6
0.4
0.5
0.2
0 0
10% FBS 5% PRPr 5% PRPr 5% PRPr 10% FBS 5% PRPr 5% PRPr 5% PRPr
(EDTA) (SC) (ACD) (EDTA) (SC) (ACD)
1.6 3
Oct-4 mRNA relative expression
SOX9 mRNA relative expression

1.4
2.5
1.2
2
1
0.8 1.5
0.6
1
0.4
0.5
0.2
0 0
10% FBS 5% PRPr 5% PRPr 5% PRPr 10% FBS 5% PRPr 5% PRPr 5% PRPr
(EDTA) (SC) (ACD) (EDTA) (SC) (ACD)

Figure 6: Relative gene expression. RUNX2, PPAR𝛾2, SOX9, and Oct-4 gene expression in cells cultured in medium supplemented with 10%
FBS (control group) or 5% PRPr obtained with EDTA, SC, or ACD. Data are expressed as relative quantification of gene expression (RQ).
Upper and lower error bars correspond to RQ maximum and RQ minimum, respectively.
Stem Cells International 9

discussion in the literature regarding the PRP effects on chon- [3] M. Sánchez, J. Azofra, E. Anitua et al., “Plasma rich in growth
drogenesis, with some groups claiming its inducing effects factors to treat an articular cartilage avulsion: a case report,”
[70, 71] and others its inhibitory effects [72, 73]. Recently, our Medicine and Science in Sports and Exercise, vol. 35, no. 10, pp.
group has showed that it can be dependent on the concentra- 1648–1652, 2003.
tion of PRPr used in cell medium, with lower concentrations [4] E. Kon, G. Filardo, B. Di Matteo, and M. Marcacci, “PRP for
inducing chondrogenesis and higher concentrations inhibit- the treatment of cartilage pathology,” The Open Orthopaedics
ing it [74]. Oct-4 expression presented an intense variability, Journal, vol. 7, pp. 120–128, 2013.
with upregulation in EDTA and ACD groups and downreg- [5] D. W. Taylor, M. Petrera, M. Hendry, and J. S. Theodoropoulos,
ulation in SC group. Whether this can be an indicator of “A systematic review of the use of platelet-rich plasma in sports
medicine as a new treatment for tendon and ligament injuries,”
the maintenance of cells stemness or not, it must be further
Clinical Journal of Sport Medicine, vol. 21, no. 4, pp. 344–352,
investigated. In general, gene expression was similar between 2011.
PRP groups, although SC was the group that presented the [6] Y. Kajikawa, T. Morihara, H. Sakamoto et al., “Platelet-rich
smaller variation compared to 10% FBS culture, evidencing plasma enhances the initial mobilization of circulation-derived
that cells presented the lightest changes in their phenotype cells for tendon healing,” Journal of Cellular Physiology, vol. 215,
with this treatment. no. 3, pp. 837–845, 2008.
[7] T. M. McCarrel, T. Minas, and L. A. Fortier, “Optimization of
leukocyte concentration in platelet-rich plasma for the treat-
5. Conclusion ment of tendinopathy,” The Journal of Bone & Joint Surgery—
The literature provides a variety of methodologies to obtain American Volume, vol. 94, no. 19, article e143, pp. 1–8, 2012.
PRP. The first variation is the methodology to collect blood [8] G. Reurink, G. J. Goudswaard, M. H. Moen et al., “Platelet-
and the anticoagulant used. In this paper, we analyzed the rich plasma injections in acute muscle injury,” The New England
Journal of Medicine, vol. 370, no. 26, pp. 2546–2547, 2014.
effects of three different anticoagulants, obtained in commer-
cially available tubes, on PRP obtaining. Although no signif- [9] J. W. Hammond, R. Y. Hinton, L. A. Curl, J. M. Muriel, and R.
M. Lovering, “Use of autologous platelet-rich plasma to treat
icant change in the amount of growth factors released was
muscle strain injuries,” The American Journal of Sports Medicine,
observed, some features could be highlighted. The blood col- vol. 37, no. 6, pp. 1135–1142, 2009.
lection in tubes containing EDTA resulted in higher platelet [10] R. E. Marx, E. R. Carlson, R. M. Eichstaedt, S. R. Schimmele, J. E.
yield in the whole blood. However, this was accompanied by Strauss, and K. R. Georgeff, “Platelet-rich plasma: growth factor
an increase of MPV following the centrifugation steps, which enhancement for bone grafts,” Oral Surgery, Oral Medicine, Oral
is an indicator of change in platelet morphology. On the other Pathology, Oral Radiology, and Endodontics, vol. 85, no. 6, pp.
hand, the use of tubes containing citrate solutions resulted 638–646, 1998.
in a greater induction of MSC proliferation. Particularly, the [11] G. Weibrich, T. Hansen, W. Kleis, R. Buch, and W. E. Hitzler,
obtaining in SC resulted in the higher platelet recovery after “Effect of platelet concentration in platelet-rich plasma on peri-
the first centrifugation step. If ACD is used, the reduction of implant bone regeneration,” Bone, vol. 34, no. 4, pp. 665–671,
the tube size may increase platelet recovery. In addition, the 2004.
PRP obtained in SC presented the smallest variation in MSC [12] A. D. Mazzocca, M. B. R. McCarthy, D. M. Chowaniec et al.,
gene expression compared to cells cultured in the presence “The positive effects of different platelet-rich plasma methods
of 10% FBS. Therefore, in order to obtain a bigger amount of on human muscle, bone, and tendon cells,” American Journal of
platelets and induce MSC proliferation without dramatically Sports Medicine, vol. 40, no. 8, pp. 1742–1749, 2012.
interfering with their phenotype, we suggest the use of SC as [13] K. Schallmoser, C. Bartmann, E. Rohde et al., “Human platelet
anticoagulant for PRP acquisition. lysate can replace fetal bovine serum for clinical-scale expan-
sion of functional mesenchymal stromal cells,” Transfusion, vol.
47, no. 8, pp. 1436–1446, 2007.
Competing Interests [14] E. Anitua, M. Sánchez, M. M. Zalduendo et al., “Fibroblastic
response to treatment with different preparations rich in growth
The authors declare no competing interests. factors,” Cell Proliferation, vol. 42, no. 2, pp. 162–170, 2009.
[15] V. K. Gonzales, E. L. W. de Mulder, T. de Boer et al., “Platelet-
rich plasma can replace fetal bovine serum in human meniscus
Acknowledgments cell cultures,” Tissue Engineering—Part C: Methods, vol. 19, no.
The authors would like to thank all the staff at Amil Life 11, pp. 892–899, 2013.
Sciences for support. [16] T. E. Foster, B. L. Puskas, B. R. Mandelbaum, M. B. Gerhardt,
and S. A. Rodeo, “Platelet-rich plasma: from basic science to
clinical applications,” American Journal of Sports Medicine, vol.
References 37, no. 11, pp. 2259–2272, 2009.
[17] M. Pakyari, A. Farrokhi, M. K. Maharlooei, and A. Ghahary,
[1] R. E. Marx, “Platelet-rich plasma (PRP): what is PRP and what “Critical role of transforming growth factor beta in different
is not PRP?” Implant Dentistry, vol. 10, no. 4, pp. 225–228, 2001. phases of wound healing,” Advances in Wound Care, vol. 2, no.
[2] G. Filardo, E. Kon, R. Buda et al., “Platelet-rich plasma intra- 5, pp. 215–224, 2013.
articular knee injections for the treatment of degenerative [18] D. I. R. Holmes and I. Zachary, “The vascular endothelial
cartilage lesions and osteoarthritis,” Knee Surgery, Sports Trau- growth factor (VEGF) family: angiogenic factors in health and
matology, Arthroscopy, vol. 19, no. 4, pp. 528–535, 2011. disease,” Genome Biology, vol. 6, no. 2, article 209, 2005.
10 Stem Cells International

[19] J. Donovan, D. Abraham, and J. Norman, “Platelet-derived [33] A. Mishra, K. Harmon, J. Woodall, and A. Vieira, “Sports
growth factor signaling in mesenchymal cells,” Frontiers in medicine applications of platelet rich plasma,” Current Pharma-
Bioscience, vol. 18, no. 1, pp. 106–119, 2013. ceutical Biotechnology, vol. 13, no. 7, pp. 1185–1195, 2012.
[20] E. Lopez-Vidriero, K. A. Goulding, D. A. Simon, D. H. John- [34] O. Bausset, L. Giraudo, J. Veran et al., “Formulation and storage
son, and M. Sanchez, “Poor standardization in platelet-rich of platelet-rich plasma homemade product,” BioResearch Open
therapies hampers advancement,” Arthroscopy: The Journal of Access, vol. 1, no. 3, pp. 115–123, 2012.
Arthroscopic and Related Surgery, vol. 26, no. 6, pp. 724–725, [35] Y. Yamada, M. Ueda, T. Naiki, M. Takahashi, K.-I. Hata, and
2010. T. Nagasaka, “Autogenous injectable bone for regeneration
[21] T. Wright-Carpenter, P. Klein, P. Schäferhoff, H. J. Appell, L. with mesenchymal stem cells and platelet-rich plasma: tissue-
M. Mir, and P. Wehling, “Treatment of muscle injuries by local engineered bone regeneration,” Tissue Engineering, vol. 10, no.
administration of autologous conditioned serum: a pilot study 5-6, pp. 955–964, 2004.
on sportsmen with muscle strains,” International Journal of [36] H. Hemeda, J. Kalz, G. Walenda, M. Lohmann, and W. Wagner,
Sports Medicine, vol. 25, no. 8, pp. 588–593, 2004. “Heparin concentration is critical for cell culture with human
[22] Z. R. Mrowiec, L. Oleksowicz, J. P. Dutcher, M. De Leon- platelet lysate,” Cytotherapy, vol. 15, no. 9, pp. 1174–1181, 2013.
Fernandez, P. Lalezari, and E. G. Puszkin, “A novel technique [37] J. Araki, M. Jona, H. Eto et al., “Optimized preparation method
for preparing improved puffy coat platelet concentrates,” Blood of platelet-concentrated plasma and noncoagulating platelet-
Cells, Molecules, and Diseases, vol. 21, no. 1, pp. 25–33, 1995. derived factor concentrates: maximization of platelet concen-
tration and removal of fibrinogen,” Tissue Engineering—Part C:
[23] S. Ferizhandy Ali, “Platelet activation in stored platelet con-
Methods, vol. 18, no. 3, pp. 176–185, 2012.
centrates: comparision of two methods preparation,” Journal of
Blood Disorders & Transfusion, vol. 2, no. 2, pp. 2–4, 2011. [38] R. A. R. Bowen and A. T. Remaley, “Interferences from blood
collection tube components on clinical chemistry assays,” Bio-
[24] G. Bernardini, F. Chellini, B. Frediani, A. Spreafico, and A.
chemia Medica, vol. 24, no. 1, pp. 31–44, 2014.
Santucci, “Human platelet releasates combined with polygly-
colic acid scaffold promote chondrocyte differentiation and [39] P. R. Amable, R. B. V. Carias, M. V. T. Teixeira et al., “Platelet-
phenotypic maintenance,” Journal of Biosciences, vol. 40, no. 1, rich plasma preparation for regenerative medicine: optimiza-
pp. 61–69, 2015. tion and quantification of cytokines and growth factors,” Stem
Cell Research and Therapy, vol. 4, no. 3, article 67, 2013.
[25] E. Kon, R. Buda, G. Filardo et al., “Platelet-rich plasma: intra-
[40] K. J. Livak and T. D. Schmittgen, “Analysis of relative gene
articular knee injections produced favorable results on degen-
expression data using real-time quantitative PCR and the 2−ΔΔ𝐶T
erative cartilage lesions,” Knee Surgery, Sports Traumatology,
method,” Methods, vol. 25, no. 4, pp. 402–408, 2001.
Arthroscopy, vol. 18, no. 4, pp. 472–479, 2010.
[41] Y. Zhu, M. Yuan, H. Y. Meng et al., “Basic science and clinical
[26] E. Anitua and G. Orive, “Short implants in maxillae and
application of platelet-rich plasma for cartilage defects and
mandibles: a retrospective study with 1 to 8 years of follow-up,”
osteoarthritis: a review,” Osteoarthritis and Cartilage, vol. 21, no.
Journal of Periodontology, vol. 81, no. 6, pp. 819–826, 2010.
11, pp. 1627–1637, 2013.
[27] E. Anitua, B. Pelacho, R. Prado et al., “Infiltration of plasma [42] E. Anitua, R. Prado, and G. Orive, “Endogenous morphogens
rich in growth factors enhances in vivo angiogenesis and and fibrin bioscaffolds for stem cell therapeutics,” Trends in
improves reperfusion and tissue remodeling after severe hind Biotechnology, vol. 31, no. 6, pp. 364–374, 2013.
limb ischemia,” Journal of Controlled Release, vol. 202, pp. 31–
[43] M. Betsch, J. Schneppendahl, S. Thuns et al., “Bone marrow
39, 2015.
aspiration concentrate and platelet rich plasma for osteochon-
[28] J.-C. Lee, H. J. Min, H. J. Park, S. Lee, S. C. Seong, and M. dral repair in a porcine osteochondral defect model,” PLoS ONE,
C. Lee, “Synovial membrane-derived mesenchymal stem cells vol. 8, no. 8, article e71602, 2013.
supported by platelet-rich plasma can repair osteochondral
[44] H. Li, A. Usas, M. Poddar et al., “Platelet-rich plasma promotes
defects in a rabbit model,” Arthroscopy, vol. 29, no. 6, pp. 1034–
the proliferation of human muscle derived progenitor cells and
1046, 2013.
maintains their stemness,” PLoS ONE, vol. 8, no. 6, Article ID
[29] M. Pihut, M. Szuta, E. Ferendiuk, and D. Zeńczak-Więckiewicz, e64923, 2013.
“Evaluation of pain regression in patients with temporo- [45] E. Castrén, T. Sillat, S. Oja et al., “Osteogenic differentiation
mandibular dysfunction treated by intra-articular platelet-rich of mesenchymal stromal cells in two-dimensional and three-
plasma injections: a preliminary report,” BioMed Research dimensional cultures without animal serum,” Stem Cell Research
International, vol. 2014, Article ID 132369, 7 pages, 2015. & Therapy, vol. 6, no. 1, article 167, pp. 1–13, 2015.
[30] N. Kakudo, N. Morimoto, S. Kushida, T. Ogawa, and K. [46] R. L. McShine, P. C. Das, C. T. Smit Sibinga, and B. Brozovic,
Kusumoto, “Platelet-rich plasma releasate promotes angiogen- “Differences between the effects of EDTA and citrate anticoag-
esis in vitro and in vivo,” Medical Molecular Morphology, vol. 47, ulants on platelet count and mean platelet volume,” Clinical and
no. 2, pp. 83–89, 2014. Laboratory Haematology, vol. 12, no. 3, pp. 277–285, 1990.
[31] H.-R. Lee, K. M. Park, Y. K. Joung, K. D. Park, and S. H. Do, [47] R. L. McShine, P. C. Das, C. T. S. Sibinga, and B. Brozović,
“Platelet-rich plasma loaded hydrogel scaffold enhances chon- “Effect of EDTA on platelet count and other platelet parameters
drogenic differentiation and maturation with up-regulation of in blood and blood components collected with CPDA-1,” Vox
CB1 and CB2,” Journal of Controlled Release, vol. 159, no. 3, pp. Sanguinis, vol. 61, no. 2, pp. 84–89, 1991.
332–337, 2012. [48] J. Araki, M. Jona, H. Eto et al., “Optimized preparation method
[32] X. Xie, Y. Wang, C. Zhao et al., “Comparative evaluation of of platelet-concentrated plasma and noncoagulating platelet-
MSCs from bone marrow and adipose tissue seeded in PRP- derived factor concentrates: maximization of platelet concen-
derived scaffold for cartilage regeneration,” Biomaterials, vol. 33, tration and removal of fibrinogen,” Tissue Engineering Part C:
no. 29, pp. 7008–7018, 2012. Methods, vol. 18, no. 3, pp. 176–185, 2012.
Stem Cells International 11

[49] J. H. Ladenson, L. M. B. Tsai, J. M. Michael, G. Kessler, and J. H. [66] E. Rubio-Azpeitia and I. Andia, “Partnership between platelet-
Joist, “Serum versus heparinized plasma for eighteen common rich plasma and mesenchymal stem cells: in vitro experience,”
chemistry tests: is serum the appropriate specimen?” American Muscles, Ligaments and Tendons Journal, vol. 4, no. 1, pp. 52–62,
Journal of Clinical Pathology, vol. 62, no. 4, pp. 545–552, 1974. 2014.
[50] P. M. W. Bath and R. J. Butterworth, “Platelet size: measurement, [67] D. Baksh, L. Song, and R. S. Tuan, “Adult mesenchymal stem
physiology and vascular disease,” Blood Coagulation and Fibri- cells: characterization, differentiation, and application in cell
nolysis, vol. 7, no. 2, pp. 157–161, 1996. and gene therapy,” Journal of Cellular and Molecular Medicine,
[51] Y. Park, N. Schoene, and W. Harris, “Mean platelet volume as an vol. 8, no. 3, pp. 301–316, 2004.
indicator of platelet activation: methodological issues,” Platelets, [68] J. Zhang and J. H.-C. Wang, “Human tendon stem cells better
vol. 13, no. 5-6, pp. 301–306, 2002. maintain their stemness in hypoxic culture conditions,” PLoS
[52] R. H. Aster, “Blood platelet kinetics and platelet transfusion,” ONE, vol. 8, no. 4, Article ID e61424, 2013.
The Journal of Clinical Investigation, vol. 123, no. 11, pp. 4564– [69] H. J. Sung, S. C. Hong, J. H. Yoo et al., “Stemness evaluation
4565, 2013. of mesenchymal stem cells from placentas according to devel-
[53] C. Oviedo and J. Rodrı́guez, “EDTA: the chelating agent under opmental stage: comparison to those from adult bone marrow,”
environmental scrutiny,” Quimica Nova, vol. 26, no. 6, pp. 901– Journal of Korean Medical Science, vol. 25, no. 10, pp. 1418–1426,
905, 2003. 2010.
[54] M. Fukaya and A. Ito, “A new economic method for prepar- [70] K. Akeda, H. S. An, M. Okuma et al., “Platelet-rich plasma stim-
ing platelet-rich plasma,” Plastic and Reconstructive Surgery— ulates porcine articular chondrocyte proliferation and matrix
Global Open, vol. 2, no. 6, article e162, 2014. biosynthesis,” Osteoarthritis and Cartilage, vol. 14, no. 12, pp.
[55] H. Lei, L. Gui, and R. Xiao, “The effect of anticoagulants on the 1272–1280, 2006.
quality and biological efficacy of platelet-rich plasma,” Clinical [71] A. Spreafico, F. Chellini, B. Frediani et al., “Biochemical inves-
Biochemistry, vol. 42, no. 13-14, pp. 1452–1460, 2009. tigation of the effects of human platelet releasates on human
[56] P. Pignatelli, F. M. Pulcinelli, F. Ciatti et al., “Acid Citrate articular chondrocytes,” Journal of Cellular Biochemistry, vol.
Dextrose (ACD) formula A as a new anticoagulant in the 108, no. 5, pp. 1153–1165, 2009.
measurement of in vitro platelet aggregation,” Journal of Clinical [72] C. Gaissmaier, J. Fritz, T. Krackhardt, I. Flesch, W. K. Aicher,
Laboratory Analysis, vol. 9, no. 2, pp. 138–140, 1995. and N. Ashammakhi, “Effect of human platelet supernatant on
[57] P. Pignatelli, F. M. Pulcinelli, F. Ciatti, M. Pesciotti, P. Ferroni, proliferation and matrix synthesis of human articular chondro-
and P. P. Gazzaniga, “Effects of storage on in vitro platelet cytes in monolayer and three-dimensional alginate cultures,”
responses: comparison of ACD and Na citrate anticoagulated Biomaterials, vol. 26, no. 14, pp. 1953–1960, 2005.
samples,” Journal of Clinical Laboratory Analysis, vol. 10, no. 3, [73] C. Kaps, A. Loch, A. Haisch et al., “Human platelet supernatant
pp. 134–139, 1996. promotes proliferation but not differentiation of articular chon-
[58] C. E. A. Jochems, J. B. F. van der Valk, F. R. Stafleu, and V. drocytes,” Medical and Biological Engineering and Computing,
Baumans, “The use of fetal bovine serum: ethical or scientific vol. 40, no. 4, pp. 485–490, 2002.
problem?” Alternatives to Laboratory Animals, vol. 30, no. 2, pp. [74] R. J. do Amaral, A. Matsiko, M. R. Tomazette et al., “Platelet-rich
219–227, 2002. plasma releasate differently stimulates cellular commitment
[59] G. Gstraunthaler, “Alternatives to the use of fetal bovine serum: toward the chondrogenic lineage according to concentration,”
serum-free cell culture,” ALTEX: Alternativen zu Tierexperi- Journal of Tissue Engineering, vol. 6, 2015.
menten, vol. 20, no. 4, pp. 275–281, 2003.
[60] E. Cordeiro-Spinetti, W. de Mello, L. S. Trindade, D. D. Taub,
R. S. Taichman, and A. Balduino, “Human bone marrow mes-
enchymal progenitors: perspectives on an optimized in vitro
manipulation,” Frontiers in Cell and Developmental Biology, vol.
2, pp. 1–8, 2014.
[61] B. L. Eppley, J. E. Woodell, and J. Higgins, “Platelet quantifi-
cation and growth factor analysis from platelet-rich plasma:
implications for wound healing,” Plastic and Reconstructive
Surgery, vol. 114, no. 6, pp. 1502–1508, 2004.
[62] G. Weibrich, W. K. G. Kleis, G. Hafner, and W. E. Hitzler,
“Growth factor levels in platelet-rich plasma and correlations
with donor age, sex, and platelet count,” Journal of Cranio-
Maxillofacial Surgery, vol. 30, no. 2, pp. 97–102, 2002.
[63] E. A. Sundman, B. J. Cole, and L. A. Fortier, “Growth factor and
catabolic cytokine concentrations are influenced by the cellular
composition of platelet-rich plasma,” American Journal of Sports
Medicine, vol. 39, no. 10, pp. 2135–2140, 2011.
[64] H. El-Sharkawy, A. Kantarci, J. Deady et al., “Platelet-rich
plasma: growth factors and pro- and anti-inflammatory proper-
ties,” Journal of Periodontology, vol. 78, no. 4, pp. 661–669, 2007.
[65] T. N. Castillo, M. A. Pouliot, Hyeon Joo Kim, and J. L. Dragoo,
“Comparison of growth factor and platelet concentration from
commercial platelet-rich plasma separation systems,” The Amer-
ican Journal of Sports Medicine, vol. 39, no. 2, pp. 266–271, 2011.
 International Journal of

Peptides

Advances in
BioMed
Research International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Stem Cells
International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Virolog y
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
International Journal of
Genomics
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Journal of
Nucleic Acids

Zoology
 International Journal of

Hindawi Publishing Corporation Hindawi Publishing Corporation

http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Submit your manuscripts at

http://www.hindawi.com

Journal of The Scientific

Signal Transduction
Hindawi Publishing Corporation
World Journal
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Genetics Anatomy International Journal of Biochemistry Advances in

Research International
Hindawi Publishing Corporation
Research International
Hindawi Publishing Corporation
Microbiology
Hindawi Publishing Corporation
Research International
Hindawi Publishing Corporation
Bioinformatics
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Enzyme International Journal of Molecular Biology Journal of

Archaea
Hindawi Publishing Corporation
Research
Hindawi Publishing Corporation
Evolutionary Biology
Hindawi Publishing Corporation
International
Hindawi Publishing Corporation
Marine Biology
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014