International Journal of

Molecular Sciences
ArticleEffect of Platelet-Rich Plasma on M1/M2 Macrophage Polarization
Ryoka Uchiyama 1,2 , Eriko Toyoda 1,2 , Miki Maehara 1,2 , Shiho Wasai 1,2 , Haruka Omura 1,2 ,Masahiko Watanabe 1,2 and Masato Sato 1,2, *12*

Citation: Uchiyama, R.; Toyoda, E.;
Maehara, M.; Wasai, S.; Omura, H.;

Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya,
Isehara, Kanagawa 259-1193, Japan; 9bmrm002@mail.u-tokai.ac.jp (R.U.); etoyoda@tokai-u.jp (E.T.);m-maehara@tsc.u-tokai.ac.jp (M.M.); s4.1x2.5s@gmail.com (S.W.); hrk.omr@gmail.com (H.O.);
masahiko@is.icc.u-tokai.ac.jp (M.W.)
Center for Musculoskeletal innovative Research and Advancement (C-MiRA), Tokai University Graduate
School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
Correspondence: sato-m@is.icc.u-tokai.ac.jp; Tel.: +81-463-93-1121; Fax: +81-463-96-4404

Abstract: Osteoarthritis of the knee (OAK) is a chronic degenerative disease and progresses with animbalance of cytokines and macrophages in the joint. Studies regarding the use of platelet-rich plasma(PRP) as a point-of-care treatment for OAK have reported on its effect on tissue repair and suppressionof inflammation but few have reported on its effect on macrophages and macrophage polarization.
Based on our clinical experience with two types of PRP kits Cellaid Serum Collection Set P type kit(leukocyte-poor-PRP) and an Autologous Protein Solution kit (APS leukocyte-rich-PRP), we investigatedthe concentrations of humoral factors in PRPs prepared from the two kits and the effect of humoral
factors on macrophage phenotypes. We found that the concentrations of cell components and humoral
factors differed between PRPs purified using the two kits; APS had a higher concentration of M1 andM2 macrophage related factors. The addition of PRP supernatants to the culture media of monocytederived macrophages and M1 polarized macrophages revealed that PRPs suppressed M1 macrophage
polarization and promoted M2 macrophage polarization. This research is the first to report the effect of
PRPs purified using commercial kits on macrophage polarization.

Watanabe, M.; Sato, M. Effect ofPlatelet-Rich Plasma on M1/M2

Keywords: platelet rich plasma; autologous protein solution; macrophage

Macrophage Polarization. Int. J. Mol.
Sci. 2021, 22, 2336. https://doi.org/
10.3390/ijms22052336

1. Introduction
Academic Editor: Malgorzata Kloc
Received: 27 January 2021
Accepted: 23 February 2021
Published: 26 February 2021

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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Osteoarthritis of the knee (OAK) is a chronic degenerative disease that damages articularcartilage. OAK is characterized by aberrant cartilage metabolism, osteophyte formation, synovial tissue thickening, and macrophage infiltration. These changes contribute to the progressof OAK, resulting in joint pain and dysfunction [1,2]. There are about 25 million patients with
OAK in Japan, and the number is expected to increase in tandem with Japan’s hyper-aging
population [3]. No treatment currently exists that lead to a complete recovery from OAK.
Conservative treatments (i.e., exercise, oral treatment, intra-articular injection of hyaluronic
acids) are performed for mild cases, and surgical treatments (i.e., arthroscopic debridement of
the knee joint, high tibial osteotomy, and total knee arthroplasty) for severe cases [4,5]. Joint
treatment with platelet-rich plasma (PRP) has recently attracted attention as a point-of-caretreatment option to bridge the gap between conservative and surgical treatments.
PRP is an autologous blood product generated by centrifuging whole blood andconcentrating platelets, containing diverse growth factors and cytokines [6]. The highconcentration of anti-inflammatory factors in PRPs may suppress inflammation, growth
factors may promote tissue repair, and in total these factors may improve symptoms [7,8].
Efficacy of platelet concentrates in promoting wound healing and tissue regeneration has
been at the center of scientific debate over the past few decades [9]. Various purification
kits have recently been marketed as this treatment has become more widespread. However,the blood cell components and humoral factors in PRPs depend on the purification method.
Leukocyte-poor PRP (LP-PRP) lacks leukocytes whereas leukocyte-rich PRP (LR-PRP) do

Int. J. Mol. Sci. 2021, 22, 2336. https://doi.org/10.3390/ijms22052336

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contain leukocytes. We have clinical experience with both types of kits: Cellaid Serum
Collection Set P type kit (LP-PRP) and an Autologous Protein Solution kit (APS, LR-PRP).
APS, with an added dehydration step using polyacrylamide beads during purification, hasbeen reported to achieve a high concentration of not only platelets and leukocytes but also
humoral factors such as IGF-1 [6]. However, there is no consensus on how differences in
the components of PRP products affect their therapeutic efficacies.
One possibility is the effect of PRPs on macrophages. Macrophages are immune
cells that play a crucial role in innate immunity and also participate in tissue repair andremodeling [10]. Macrophages can be polarized into two phenotypes in response to stimuli
from their microenvironment. Classically activated macrophages (M1 macrophages) are
induced by, for example, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α),and bacterial-derived lipopolysaccharides (LPS), and are known to release inflammatory
cytokines that promote tissue damage and inflammation [11–13]. Alternatively activatedmacrophages (M2 macrophages) are induced by, for example, interleukin-4 (IL-4) and IL-13,
and are known to release anti-inflammatory cytokines that promote tissue remodeling andsuppress inflammation [14,15].
Recent studies have shown that M1 macrophages are present in the synovium andsynovial fluid in OAK patients and involved in disease progression, suggesting they may betargeted for treatment [16–19]. Many studies have reported PRPs suppressing inflammationand inducing articular cartilage repair, but few studies have reported the effect of PRPs on
macrophage phenotypes [20–22].
In this study, LP-PRP and APS were purified from peripheral blood of healthy subjects
using two PRP preparation kits in which we have clinical experience. We then compared theconcentrations of humoral factors related to M1/M2 macrophage polarization in the respective
PRPs, and then added the respective PRP supernatants to the culture media of monocytederived macrophages (MDMs) to investigate their effect on macrophage phenotypes.
2. Results
2.1. Hematological Analysis of PRPs
From each healthy donor, 120 mL of peripheral blood was collected and 60 mL per kit
was used to prepare both LP-PRP and APS. Immediately after preparation, hematologicalanalysis was performed for whole blood, LP-PRP, and APS (Figure 1A). Erythrocyte
concentration was negligible for LP-PRP while it was lower for APS compared to whole
blood. Leukocyte concentration was negligible for LP-PRP while it was higher for APS
compared to whole blood. Platelet concentrations were higher for both LP-PRP and
APS compared to whole blood and did not significantly differ between LP-PRP and APS(Figure 1B). A comparison of the ratio of leukocyte types contained in whole blood and
APS revealed that in APS, the ratios of neutrophils and eosinophils were higher while theratios of monocytes and lymphocytes were lower (Figure 1C). According to the coding
system presented by Kon et. al., the PRP codes for LP-PRP and APS are 210-00-00 and
214-15-10, respectively [23].

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Figure1.1.Purification
Purificationand
andanalyses
analysesofofplatelet-richplatelet-richplasmas
plasmas(PRPs)
from
two
kits.
Flow
PRP
purification
analFigure(PRPs)
from
two
kits.
(A)(A)
Flow
of of
PRP
purification
andandanalyses.
yses. each
Fromofeach
12 healthy
subjects,
120of peripheral
was collected
andadded
was added
to 12ofmLof anticoFrom
the of12thehealthysubjects,
120 mL
ofmL
peripheral
bloodblood
was collected
and wasto 12 mL
anticoagulant
agulant citrate-dextrose
Leukocyte-poor
PRP (LP-PRP)
and Autologous
Protein (APS)
Solution(APS)
wereusingpurified
citrate-dextrose
solution. solution.
Leukocyte-poor
PRP (LP-PRP)
and Autologous
Protein Solutionwere
purified
their
using
their
respective
kits,
and
hematologicalanalysiswasperformed
immediately
following
purification.
Humoralrespective kits, and hematological analysis was performed immediately following purification. Humoral factor analysis wasfactor analysis was performed with frozen stored samples for various macrophage related factors. (B) Comparison of
performed with frozen stored samples for various macrophage related factors. (B) Comparison of leukocyte, erythrocyte,
leukocyte, erythrocyte, and platelet concentrations in LP-PRP and APS. (C) Comparison of the ratios of leukocyte types
and platelet concentrations in LP-PRP and APS. (C) Comparison of the ratios of leukocyte types in whole blood and APS.
in whole blood and APS. n = 12 PRP donors for each kit. * p < 0.05, ** p < 0.01.
n = 12 PRP donors for each kit. * p < 0.05, ** p < 0.01.

2.2. Analysis of Humoral Factors
2.2. Analysis of Humoral Factors
We investigated the effect of PRPs on macrophage polarization by analyzing the
We investigatedeffect of PRPs.
PRPs Macrophages
on macrophage
polarization
by analyzing
thehumoralfactors in thethe
respective
respondto factors
such as C-X-C
humoralfactorsintherespective
PRPs.
Macrophages
respondtofactors
such
as
C-X-C
motif chemokine ligand 9 (CXCL9), interferon gamma-induced protein 10 (IP-10),
motif
chemokine
ligand 9 (CXCL9),gamma-induced
protein
10 (IP-10), C-XXX-C
C-XXX-C
motif chemokine
ligand 1interferon(CX3CL1),
and macrophage
chemoattractant
proteinmotif
chemokine
ligand1(CX3CL1),
andmacrophage
chemoattractant
protein 1 (MCP-1),
1 (MCP-1), and are thereby recruited to areas of inflammation. M1 macrophages
respondandare aretherebyrecruited
to areas of inflammation.
macrophages respondto and are
to andpolarized
by granulocyte
macrophage M1
colony-stimulating
factor (GM-CSF),
polarizedbygranulocyte
macrophagecolony-stimulating
factor(GM-CSF),
TNF-α,
IFN-γ,
TNF-α, IFN-γ, while M2 macrophages respond to and are polarized by macrophage
while M2 macrophages respond to and are polarized by macrophage colony-stimulating

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colony-stimulating
factor(M-CSF),
IL-4,receptor
IL-10, IL-13,
IL-1 receptor
antagonist(IL-1RA),factor (M-CSF), IL-4,
IL-10,
IL-13, IL-1
antagonist(IL-1RA),and transforming
andtransforming
growthfactor-beta(TGF-β)[14].
Asaresultofquantitative
growth factor-beta (TGF-β) [14]. As a result of quantitative analysis, both LP-PRP analysis,
and APSboth
LP-PRP
and
APS
contained
all of these
A comparisonof theconcentrations
contained
all ofthese
factors.
A comparison
offactors.
the concentrationsof thesefactors
in LP-PRP
offactors thatin theLP-PRP
and APS
showed that the concentration
macroandthese
APS showed
concentrationof macrophage-recruitment
chemokineofIP-10
andphage-recruitment
chemokine
IP-10
and
CX3CL1wassignificantly
higherin
APS
CX3CL1 was significantly higher in APS (Figure 2A). Moreover, the concentrations of(Figthe
ure
2A).
Moreover,theconcentrations
of
the
M1
macrophage
relatedfactor
TNF-α(FigM1 macrophage related factor TNF-α (Figure 2B) and the M2 macrophage related factors
ure2B) IL-10,
and theM2 macrophage
M-CSF,
IL-10, IL-1RA,
andinTGF-β(FigM-CSF,
IL-1RA,and TGF-β related(Figure factors2C) werealso significantly
higherAPS. These
ure
2C)
were
also
significantly
higher
in
APS.
These
results
showedthat
LP-PRPandresults showed that LP-PRP and APS differ in the concentrations of cytokines involved
APS
differintheconcentrationsofcytokines
involvedinmacrophage
polarization
within macrophage polarization with higher concentrations of both M1 and M2 macrophagehigherrelatedconcentrations
factors in APS.of both M1 and M2 macrophage related factors in APS.

Figure 2. Analysis
Analysis ofof humoral
humoral factors
factors inin PRPs. Comparison
Comparison ofof concentrations
concentrationsofof eacheach factor
factor inin LP-PRP
LP-PRP andand APS.
APS. (A)(A)
Macrophage-recruitment factors(C)
M2
macrophage
relatedfactors.
n =n12= PRP
donorsMacrophage-recruitment
factors(B)(B)M1
M1macrophage
macrophagerelated
relatedfactors
factors(C)
M2
macrophage
relatedfactors.
12 PRP
donorsfor eachp < 0.05,
p < 0.01.
for eachkit. * kit.
p < *0.05,** p <**0.01.

2.3. Effect of the Addition of PRP Supernatants on Macrophage Phenotypes

Int. J. Mol. Sci. 2021, 22, 2336

5 of 162.3. Effect of the Addition of PRP Supernatants on Macrophage Phenotypes
Next, we investigated whether PRPs affect macrophage phenotypes and whether
the effect differs depending on the purification kit. M1 macrophages express IL-1β, IL-6,
and TNF-α, while M2 macrophages express MRC1, IL-10, and TGF-β [14]. Monocytes
were isolated from human peripheral blood and cultured in M-CSF for six days, thensupernatants from each PRP was added to the culture media, and the cells were cultured
for another two days. Then, the effect of PRP supernatants on the expression of the M1/M2
macrophage marker genes was confirmed by quantitative reverse transcription-polymerase
chain reaction (qRT-PCR) (Figure 3A). MDM control group served as negative control, andM1 and M2 polarized groups served as positive controls. The expression of IL-1β and
TNF-α, which are M1 macrophage markers, was lower in the LP-PRP- and APS-addedgroups compared with the MDM control group, and the expression of IL-6 also tended
to be lower. However, there was no significant difference in gene expression between the
addition of PRP supernatants purified using the two kits (Figure 3B). The expressionofInt. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
5 of 17
MRC1, which is an M2 macrophage marker, was higher in the LP-PRP- and APS-addedgroups compared with the MDM control group. The expression of IL-10 was higher in the
APS-added group compared with the MDM control group. The expression of TGF-β wasinvestigated whether PRPs affect macrophage phenotypes and whether
higher Next,
in the we
LP-PRP-addedgroup compared with the APS-added group, but no difference
the effect differs depending on the purification kit. M1 macrophages express IL-1β, IL-6,
was detected between LP-PRP and APS-added groups compared with the MDM control
and TNF-α, while M2 macrophages express MRC1, IL-10, and TGF-β [14]. Monocytes
group (Figure 3C).
were isolated from human peripheral blood and cultured in M-CSF for six days, then
Similar verification experiments were performed for cell surface markers using flow
supernatants from each PRP was added to the culture media, and the cells were cultured
cytometry. M1 macrophages express CD80 and CD86, while M2 macrophages express
for another two days. Then, the effect of PRP supernatants on the expression of theCD163 and CD206 on the cell surface [14]. A typical histogram is shown in Figure 4A.
M1/M2 macrophage marker genes was confirmed by quantitative reverse transcripThevalues were calculated and compared based on difference of the mean fluorescencetion-polymerase chain reaction (qRT-PCR) (Figure 3A). MDM control group served asintensities
the M1antibody
andisotype groups
control.served
The expression
CD80 andnegative between
control, andand M2polarized
as positiveof
controls.
TheCD86,
exwhichare
M1
macrophage
markers,
decreasedinthe
LP-PRPand
APS-addedgroupspression of IL-1β and TNF-α, which are M1 macrophage markers, was lower inthecomparedthe MDM group,no difference
observed
betweentheandpurification
LP-PRP- withand APS-addedgroupsbut
comparedwith wasthe MDM
controlgroup,
the exkits(Figure
4B).
On
the
other
hand,
there
was
no
difference
intheexpressionof theinM2pression of IL-6 also tended to be lower. However, there was no significant difference
macrophage
markers
CD163
and
CD206
betweenthe
MDM
control
group
and
the
LP-PRPgene expression between the addition of PRP supernatants purified using the two kitsor(Figure
APS-addedgroups(Figureof4C).
3B). The
expression
MRC1, which is an M2 macrophage marker, was higher inresults
that theadditionof PRP
supernatants
the expression
theThese
LP-PRPandshowed
APS-addedgroupscomparedwith
the MDM decreased
control group.
The exof pression
M1 macrophage
markers,but
was no difference
between withthe purification
kits. Onof IL-10 was
higherin there
the APS-addedgroup compared
the MDM control
thegroup.
other The
hand,
the expressionof M2
surfacemarkers tended
to be maintained
expressionof TGF-β
wasmacrophagehigher in the
LP-PRP-addedgroup compared
with
orthe
increasedby theaddition
PRP supernatants
while
the gene
expressionof IL-10
APS-addedgroup,
but noof
difference
was detected
between
LP-PRP
and APS-added
increasedin the APS-addedgroupcontrol
and TGF-β
in the LP-PRP-added group.
groups comparedwith the MDM
groupincreased(Figure 3C).

Figure 3. Cont.

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Figure3. Effectof PRP
supernatants
gene
expressionof M1/M2
macrophage
markers.
(A)
Summaryofof
thisexperiFigure
3. Effectof PRP
supernatants
on on
gene
expressionof M1/M2
macrophage
markers.
(A)
Summary
thisexperiment.
ment.
CD14 + monocytes
were isolated
from peripheral
blooddensity
using density
gradient
centrifugationand magnetic
beads.
CD14+ monocytes
were isolated
from peripheral
blood using
gradient
centrifugationand magnetic
beads.
MonoMonocytes were cultured in basal medium supplemented with 10% FBS containing M-CSF at
37 °C under 5% CO2 for six◦
cytes were cultured in basal medium supplemented with 10% FBS containing M-CSF at 37 C under 5% CO2 for six days,
days, then the medium was replaced by fresh basal medium supplemented with 10% FBS containing supernatants obthen the medium was replaced by fresh basal medium supplemented with 10% FBS containing supernatants obtained fromtained from LP-PRP or APS, and the cells were cultured for another two days. (B) Gene expression of M1 macrophage
LP-PRPor APS,
andIL-6,
the cells
were
culturedanother twodays. (B)Gene IL-10,
expressionof M1
macrophage
markers(IL-1β,markers(IL-1β,TNF-α)and(C) M2for
macrophage
markers(MRC1,
TGF-β).
Data
were analyzed
throughIL-6,
TNF-α)and(C)
M2
macrophage
markers(MRC1,
IL-10,
TGF-β).
Data
wereanalyzedthroughqRT-PCR.
-∆∆Ct
valuesqRT-PCR. -ΔΔCt values were calculated using GAPDH as an internal control. Monocyte-derived macrophages (MDM)
controlgroupusing
servedGAPDH
as negative
control,andM1 andMonocyte-derived
M2 polarized groups
served as positive
MDM control
were
calculated
as aninternal
control.
macrophages(MDM)controls.
control groupserved asgroup:control,
6 monocyte
donors,
5 experiments,
total n served= 30; LP-PRPand APS-addedgroups:6 monocyte
12 PRPdo- 5
negativeand M1and M2polarized groups
as positive
controls. MDM
controlgroup: 6donors,
monocyte
donors,nors, n = 72 group; M1 and M2 polarized groups: 6 monocyte donors, 2 or 3 experiments, n = 14 per group. * p < 0.05, ** pexperiments, total n = 30; LP-PRP- and APS-added groups: 6 monocyte donors, 12 PRP donors, n = 72 group; M1 and M2< 0.01.
polarized groups: 6 monocyte donors, 2 or 3 experiments, n = 14 per group. * p < 0.05, ** p < 0.01.

Similar verification experiments were performed for cell surface markers using flow
cytometry. M1 macrophages express CD80 and CD86, while M2 macrophages express
CD163 and CD206 on the cell surface [14]. A typical histogram is shown in Figure 4A.
The values were calculated and compared based on difference of the mean fluorescenceintensities between the antibody and isotype control. The expression of CD80 and CD86,
which are M1 macrophage markers, decreased in the LP-PRP- and APS-added groups
compared with the MDM group, but no difference was observed between the purification kits (Figure 4B). On the other hand, there was no difference in the expression of theM2 macrophage markers CD163 and CD206 between the MDM control group and the
LP-PRP- or APS-added groups (Figure 4C).
These results showed that the addition of PRP supernatants decreased the expression of M1 macrophage markers, but there was no difference between the purification
kits. On the other hand, the expression of M2 macrophage surface markers tended to be
maintained or increased by the addition of PRP supernatants while the gene expression

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Int. J. Mol. Sci. 2021, 22, 2336

7 of 16of IL-10 increased in the APS-added group and TGF-β increased in the LP-PRP-addedgroup.

Figure 4. Flow cytometric analysis of M1/M2 macrophage cell surface markers. Cells were cultured under the same proFigure
4. Flow cytometric analysis of M1/M2 macrophage cell surface markers. Cells were cultured
tocol as Figure 3. (A) A histogram representation of typical flow cytometry results. Gray and dashed lines: isotype control;thebluesameand solidlines: signals
each antibody.
Meanfluorescencerepresentation
intensity (MFI) values
each antibody
used
under
protocolas forFigure3. (A) A
histogramof oftypicalflow werecytometry
results.
to calculate ΔMFI values: ΔMFI = MFI Sample—MFI Isotype. (B) M1 macrophage markers. (C) M2 macrophage markers.
Gray and dashed lines: isotype control; blue and solid lines: signals for each antibody. Mean fluorescenceintensity (MFI) values of each antibody were used to calculate ∆MFI values: ∆MFI = MFI Sample—MFI
Isotype. (B) M1 macrophage markers. (C) M2 macrophage markers. MDM control group served as
negative control, and M1 and M2 polarized groups served as positive controls. MDM control group: 6
monocyte donors, 5 experiments, n = 30; LP-PRP- and APS-added groups: 6 monocyte donors, 12 PRP
donors, n = 72 per group; M1 and M2 polarized groups: 6 monocyte donors, 2 or 3 experiments, n = 14
per group. * p < 0.05, ** p < 0.01.

2.4. Effect of PRPs on M1 Macrophages
M1/M2 macrophages can change their phenotypes once polarized [24], and, thus,
we investigated whether PRPs can induce the polarization of M1 macrophages to M2
macrophages. Monocytes were isolated from human peripheral blood and cultured in
M-CSF for six days. MDMs were polarized to M1 macrophages and cultured for two days,
then each PRP was added to the culture medium, and the cells were cultured for anothertwo days (Figure 5A). MDM control and M1 polarized groups served as negative controls;
M1-M2 and M2 polarized groups served as positive controls. The expressions of IL-1β and
IL-6, which are M1 macrophage markers, decreased in the LP-PRP- and APS-added groups,
and suppression was greater in the APS-added group than in the LP-PRP-added group(Figure 5B). Expression of the M2 macrophage marker MRC1 increased by the addition

Int. J. Mol. Sci. 2021, 22, 2336

M1/M2 macrophages can change their phenotypes once polarized [24], and, thus,
we investigated whether PRPs can induce the polarization of M1 macrophages to M2
macrophages. Monocytes were isolated from human peripheral blood and cultured in
M-CSF for six days. MDMs were polarized to M1 macrophages and cultured for twodays, then each PRP was added to the culture medium, and the cells were cultured foranother two days (Figure 5A). MDM control and M1 polarized groups served as nega8 of 16
tive controls; M1-M2 and M2 polarized groups served as positive controls. The expressions of IL-1β and IL-6, which are M1 macrophage markers, decreased in the LP-PRPand APS-added groups, and suppression was greater in the APS-added group than in
the LP-PRP-added group (Figure 5B). Expression of the M2 macrophage marker MRC1of PRPs
and thisincreasegreaterin the
APS-added
group
in thegroup
LP-PRP-added
increasedby theadditionwasof PRPs
and thisincrease
was greaterin the than
APS-addedgroup.
The
expression
of
TGF-β
was
higherinthe
LP-PRP-added
group
than
than in the LP-PRP-added group. The expression of TGF-β was higher in thein the APSLP-PRP-added
group
than
in theresults
APS-added
group(Figureresults showedaddedgroup (Figure
5C).
These
showedthat
PRPs5C).
canThese
repolarize
M1 macrophages
PRPs can repolarize M1 macrophages to M2 macrophages.
to M2thatmacrophages.

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9 of 17

Figureof5.PRPs
Effect onof PRPs
on M1 macrophages.
(A) After
CD14+ monocyteswere
were isolated
isolated bymethodas deFigure 5. EffectM1 macrophages.
(A) After
CD14+ monocytes
bythethesamesame
methodas described
scribed in Figure 3, they were cultured in a basal medium supplemented with 10% FBS containing M-CSF at 37 °C
under◦in Figure 3,5%
they
were
culturedinabasal
mediumsupplemented
with
10%
FBS
containing
M-CSF
at
37
C
under
CO2. After six days, the media was replaced by fresh basal medium supplemented with 10% FBS containing IFN-γ + 5% CO2 .
LPS, theand media
the cellswas
werereplaced
cultured fordays
to polarize
them to M1 macrophages.
was removed,
After six days,
byanother
fresh twobasal
mediumsupplemented
with 10% The
FBSmedium
containing
IFN-γ + LPS, andthe basal medium supplemented with 10% FBS containing supernatants obtained from LP-PRP or APS was added, andthe cells were
cultured for another two days to polarize them to M1 macrophages. The medium was removed, the basal
the cells were cultured for another two days. (B) Expression of M1 macrophage markers (IL-1β, IL-6, TNF-α). (C) Exmedium supplemented
with 10% FBS
containing
supernatants
fromthrough
LP-PRPor APS-ΔΔCt
was values
added,and the cells
pression of M2 macrophage
markers(MRC1, IL-10,
TGF-β). Dataobtained
were analyzed
qRT-PCR.
were
calculated
using
GAPDH
as
an
internal
control.
MDM
controlandM1polarized
groupsservedasnegative
controls;
were cultured for another two days. (B) Expression of M1 macrophage markers (IL-1β, IL-6, TNF-α). (C) Expression of
M1-M2 and M2 polarized groups served as positive controls. MDM control, M1 polarized, M1-M2 polarized, M2 polarM2 macrophage markers (MRC1, IL-10, TGF-β). Data were analyzed through qRT-PCR. -∆∆Ct values were calculated
ized groups: 6 monocyte donors, 1 experiment, n = 6 per group; LP-PRP- and APS-added groups: 6 monocyte donors, 12
using GAPDH
as an internal
control.
MDM
PRP donors,
n = 72 per group.
* p <0.05,** control
p <0.01. and M1 polarized groups served as negative controls; M1-M2 andM2 polarized groups served as positive controls. MDM control, M1 polarized, M1-M2 polarized, M2 polarized groups:
6 monocyte donors, 1 experiment,3.nDiscussion= 6 per group; LP-PRP- and APS-added groups: 6 monocyte donors, 12 PRP donors,
n = 72 per group. * p <0.05, ** p <0.01. Platelets play an important role in the process of hemostasis and tissue repair bygathering at injury sites and secreting various cytokines and growth factors to initiate
the repair process [25]. The use of PRPs is based on the hypothesis that they eliminate
the imbalance of cytokines in joints following the injection of large amounts of platelets,
which release anti-inflammatory cytokines and growth factors [26]. Many studies havebeen published regarding humoral factors in PRPs and their effects [27–29], and we havealso previously reported that the concentrations of humoral factors differ between PRPs
derived from healthy subjects and OAK patients [27]. However, only a few studies havereported on the effect of PRPs on macrophages and their polarizations.
Macrophages are present in all tissues, play an important role in innate immunity,

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3. Discussion
Platelets play an important role in the process of hemostasis and tissue repair bygathering at injury sites and secreting various cytokines and growth factors to initiate
the repair process [25]. The use of PRPs is based on the hypothesis that they eliminate
the imbalance of cytokines in joints following the injection of large amounts of platelets,
which release anti-inflammatory cytokines and growth factors [26]. Many studies havebeen published regarding humoral factors in PRPs and their effects [27–29], and we havealso previously reported that the concentrations of humoral factors differ between PRPs
derived from healthy subjects and OAK patients [27]. However, only a few studies havereported on the effect of PRPs on macrophages and their polarizations.
Macrophages are present in all tissues, play an important role in innate immunity, andare essential for early tissue repair of damaged or inflamed areas [30,31]. Macrophages
respond to IP-10, MCP-1, CXCL9, and CX3CL1, infiltrate damaged or inflamed areas,and polarize into two functionally different types (M1 and M2) depending on their microenvironment. M1 macrophages are stimulated by IFN-γ, TNF-α, and LPS and releasepro-inflammatory factors such as IL-1β and TNF-α. M2 macrophages are stimulated byIL-4 and IL-13 and release anti-inflammatory factors such as IL-10 and growth factors suchas TGF-β, which are known to suppress inflammation and induce tissue repair [32–34].
Inflammation occurs in most synovium of OAK patients, and pathology progresses with
an imbalance of M1/M2 macrophages in the synovium and synovial fluid caused by anincrease in M1 macrophages and a decrease in M2 macrophages [17,35,36].
Therefore, the purpose of this study was to compare in PRPs derived from two
clinically relevant kits the concentrations of typical cytokines and growth factors associatedwith M1/M2 macrophages and verify the effect of PRP supernatants on macrophage
polarization. We first investigated the blood components and humoral factors in LP-PRP
and APS, which we have clinical experience administering to OAK patients [27]. We first
confirmed that compared to whole blood, APS contained higher levels of leukocytes, while
LP-PRP was leukocyte poor as expected.
Analysis of humoral factors in LP-PRP and APS showed that each contained different
concentrations of humoral factors, among which both M1 and M2 macrophage related factors were found to be at a higher concentration in APS. APS is prepared with a dehydration
process using polyacrylamide beads, which is reported to result in higher concentrationsof humoral factors [37]. In fact, APS contained a higher concentration of M2 macrophagerelated factors such as IL-10, an anti-inflammatory factor, and TGF-β, a growth factor.
At the same time, M1 macrophage-related factors such as TNF-α, a pro-inflammatory
factor, were contained at a higher concentration in APS than in LP-PRP. However, previous
reports have shown that the effects of pro-inflammatory factors are negated by the higher
concentrations of anti-inflammatory factors [38]. Moreover, an explanation for the fact thatthe concentrations of TGF-β and IL-1RA were negligible for LP-PRP is that platelets were
not activated by external factors prior to quantitative analysis.
To investigate the effect of PRPs on M1/M2 macrophage polarization, PRP supernatants were added to the culture media of monocyte-derived macrophages. Specifically,
blood components were removed through centrifugation to prevent addition of monocytes derived from PRPs and to remove the effect of leukocytes contained in PRPs. The
addition of PRP supernatants to macrophages resulted in the reduction of the expressionof M1 macrophage markers. Furthermore, when PRP supernatants were added to theculture media of macrophages, they suppressed the M1 polarization of monocyte-derived
macrophages (Figure 3B, Figure 4B) and promoted the polarization of monocyte–derived
M1 macrophages to M2 macrophages (Figure 5). The effect of PRP supernatants on themacrophage phenotype observed in this study (Figure 4B,C) suggest that PRPs have little effect on M1 macrophage related factors that may polarize macrophages to M1 macrophages.
These results indicate that PRPs may improve symptoms in OAK patients by polarizing
M1 macrophages in joints to M2 macrophages.

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In addition, reports have suggested that M2 macrophages can be categorized into threesubsets: M2a macrophages induced by IL-4 and IL-13, expressing MRC1 and IL-10; M2b induced by signals from the immune complex, expressing IL-10 and major histocompatibility
complex class II; and M2c induced by IL-10 and glucocorticoids, expressing MRC1, IL-10,
and TGF-β [32–34]. Our results suggest that LP-PRP promotes the polarization to M2cmacrophages and that APS specifically promotes the polarization to M2a macrophages.
M2a macrophages are mainly related to anti-inflammatory activity while M2c macrophages
to tissue repair [14,39]. As such, the mechanism of action of LP-PRP and APS may differ inthe elimination of the imbalance of M1/M2 macrophages in the joint.
PRPs have also been shown to enhance macrophage infiltration into tissues in tendon
repair [20]. Furthermore, it is possible that monocytes contained in APS itself may differentiate and polarize to M2 macrophages once administered to the joint. Taken together,administration of PRPs to the knee joint of OAK patients may help eliminate the imbalanceof M1/M2 macrophages in several ways: polarizing M1 macrophages already present in
the joints to M2 macrophages; populating the joint with macrophages from the surroundingarea and polarizing them to M2 macrophages; and populating the joint with monocytes
contained PRPs and polarizing them to M2 macrophages.
This study has some limitations. First, because of the difficulty in recruitment, PRPs
and monocytes used in this study were derived from peripheral blood of healthy subjects
and not OAK patients. Second, we removed cellular components through centrifugationin experiments involving the addition of PRP supernatants. In addition to cytokinesand growth factors, APS contains high concentrations of leukocytes, which may affectmacrophage polarization, and their effect when administered to the joint must be further
elucidated. For example, TNF-R released from leukocytes has been reported to inhibit thepolarization to M1 macrophages [37]. Third, 60 mL of peripheral blood provide about 6
mL of LP-PRP or 2.5 mL of APS. Thus, the clinically relevant amount of PRPs used for
intra-articular injection would result in different doses whereas equal amounts of LP-PRP
or APS supernatants were added to the media in the macrophage experiments. Fourth, instandard treatment, PRPs are used immediately after purification whereas here they werefrozen once.
This study is the first report on the effect of clinically relevant PRPs on macrophages
and macrophage phenotypes. Our findings suggest that PRPs may help improve symptoms
via modulation of macrophages in the joint, and warrant a further investigation of theireffect on macrophages as a possible mechanism of action by which PRPs promote the tissue
repair process.
4. Materials and Methods
4.1. Ethics Statement
This study was reviewed and approved by the Institutional Review Board of the Tokai
University School of Medicine (18R-134) and was conducted in compliance with relevant
guidelines. Written informed consent was obtained from all participants.
4.2. PRP Purification
To purify PRPs, 120 mL of peripheral blood collected from each of the 12 healthy
subjects (M = 5, F = 7, Age = 38.6 ± 11.0 years) was added to 12 mL of anticoagulant
citrate-dextrose solution A (ACD-A; TERUMO, Tokyo, Japan) using two 60 mL syringes.
Excess peripheral blood was centrifuged at 2200× g for 10 min and the top plasma layer
was collected and stored at −80 ◦ C until use.
LP-PRP was purified using a Cellaid Serum Collection Set P type kit (JMS, Hiroshima,
Japan). This kit consists of primary and secondary containers connected at the top by multiple tubes. Blood (20 mL) containing ACD-A was injected into the primary container andcentrifuged at 200× g for 15 min. The plasma layer containing platelets was transferred tothe secondary container via a tube at the top and centrifuged at 1200× g for 15 min. Excess
plasma was returned to the primary container, the pelletized platelets were disrupted by

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tapping, and then 2 mL of LP-PRP was collected. Approximately 6 mL of LP-PRP wascollected from 60 mL of peripheral blood using 3 kits. One hundred micro liters was used
immediately after for hematological analysis.
APS was purified using an Autologous Protein Solution (APS) kit (Zimmer Biomet,
Warsaw, IN, USA). This kit consists of two independent tubes (a GPS3 III system and an
APS Separator). Blood (60 mL) containing ACD-A was injected into the cell separation
tube (GPS III system) and centrifuged at 745× g for 15 min using a dedicated centrifuge(Zimmer Biomet) and 6 mL of the upper layer (PRP layer) was collected. This 6 mL wasadded to the APS Separator and centrifuged at 219× g for 2 min in the same centrifuge
and approximately 2.5 mL of APS was collected. One hundred micro liters was used
immediately after for hematological analysis. The collected PRPs were stored at −80 ◦ C
until use.
4.3. Hematological Analysis
The leukocyte, erythrocyte, and platelet concentrations of whole-blood, LP-PRP, and
APS samples and leukocyte compositions of whole-blood and APS samples were determined using an automated hematology analyzer (XT-1800i; Sysmex, Kobe, Japan) immediately after preparation.
4.4. Analysis of Humoral Factors
The concentrations of humoral factors in plasma, LP-PRP, and APS were measured
using a flow cytometry bead-based immunoassay (LEGENDplex™ Custom Human 13plex panel, BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol.
Plasma, LP-PRP, and APS were centrifuged at 16139× g at 4 ◦ C for 5 min to remove cellularcomponents and the supernatants were analyzed without any external activation. GMCSF, M-CSF, IFN-γ, IL-4, IL-10, IL-13, IL-1RA, free active TGF-β1, TNF-α, MCP-1, IP-10,
CXCL9, CXCL10, and CX3CL1 were measured simultaneously. Data were acquired using a
FACS Verse™ Flow Cytometer (BD Bioscience, San Diego, CA, USA) and analyzed using
BioLegend’s cloud-based LEGENDplex™ Data Analysis Software.
4.5. Isolation of Monocytes
Peripheral blood mononuclear cells (PBMCs) were separated from the buffy-coatof six healthy donors (M = 3, F = 3, Age = 32.0 ± 1.7 years) using a density gradient(Histopaque 1077, Sigma-Aldrich, St. Louis, MO, USA). PBMCs were washed with wash
buffer consisting of Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Waltham, MA,
USA) and 1% bovine serum albumin (BSA; Sigma-Aldrich) and centrifuged at 538× g at
4 ◦ C for 5 min. Contaminating red blood cells were hemolyzed with red blood cell lysing
buffer (Sigma-Aldrich) for 10 min at 37 ◦ C. Wash buffer was added and the cell suspension
was centrifuged at 538× g at 4 ◦ C for 5 min. The cell pellet was disrupted, FcR blocking
reagent (Miltenyi Biotech, Bergisch, Gladbach, Germany) was added, and the mixture was
kept at room temperature for 15 min to inhibit Fc receptor-mediated nonspecific antibody
binding. Cells were stained with mouse anti-human CD14-allophycocyanin (APC) (BD
Bioscience) at 4 ◦ C for 30 min. After washing with wash buffer, the anti-APC microbeads(Miltenyi Biotech) were bound to cells at 4 ◦ C for 20 min. The cells were washed withwash buffer and the CD14 + monocytes were isolated using an autoMACS Pro Separator(Miltenyi Biotech). Collected cells were counted with a particle counter (Sysmex).
4.6. Cell Culture
A modified version of a previously reported protocol was used [40–42].
The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 media containing GlutaMAXTM supplement (Gibco) supplemented with heat-inactivated 10% fetal
bovine serum (AusGeneX, Molendinar, Australia) and 1% penicillin-streptomycin (FujiFilm,
Tokyo, Japan) (hereinafter called basal medium).

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4.6.1. Preparation of Monocyte-Derived Macrophages and Addition of PRP Supernatants
A summary of the process is shown in Figure 3A. Isolated monocytes were seeded ata density of 1 × 105 cells/cm2 on an Upcell Multi 24 well plate (CellSeed, Tokyo, Japan)
containing basal medium supplemented with 20 ng/mL M-CSF (Peprotech, Rocky Hill,
NJ, USA), and incubated at 37 ◦ C, 5% CO2 . After six days, the medium was replacedwith medium with each of the following supplements: monocyte-derived macrophages(MDM control group)—basal medium; M1 polarized group—basal medium + 50 ng/mL
IFN-γ (Peprotech) + 100 ng/mL lipopolysaccharides from Escherichia coli (LPS, SigmaAldrich); M2 polarized group—basal medium + 20 ng/mL IL-4 (Peprotech); LP-PRP-addedgroup—basal medium + 10% LP-PRP; APS-added group—basal medium + 10% APS. PRPs
were centrifuged at 16,139× g at 4 ◦ C for 5 min to remove cellular components and thesupernatants were used. The cells were cultured for another two days and then used foranalysis. MDM control group: 6 monocyte donors, 5 experiments, total n = 30; LP-PRPand APS-added groups: 6 monocyte donors, 12 PRP donors, n = 72 group; M1 and M2
polarized groups: 6 monocyte donors, 2 or 3 experiments, n = 14 per group.
4.6.2. Preparation of M1 Macrophages and Addition of PRP Supernatants
A summary of the process is shown in Figure 5A. Isolated monocytes were seeded ata density of 1 × 105 cells/cm2 on a 96 well plate (Thermo Fisher Scientific, Tokyo, Japan)
with basal medium supplemented with 20 ng/mL M-CSF and incubated at 37 ◦ C, 5% CO2 .
After six days, the medium was removed. Basal medium was added to MDM control
group, and basal medium + 20 ng/mL IL-4 was added to M2 polarized group. Basal medium+ 50 ng/mL IFN-γ + 100 ng/mL LPS was added to the other groups. All cells were cultured
for two days, and then the medium was replaced by the following: MDM control, M2
polarized, and M1polarized groups—basal medium; M1-M2 polarized group—basal medium+ 20 ng/mL IL-4; LP-PRP-added group—basal medium + 10% LP-PRP; and APS-addedgroup—basal medium + 10% APS. PRPs were centrifuged at 16,139× g at 4 ◦ C for 5 min toremove cellular components and the supernatants were used. After two days, the medium
was removed, the cells were washed with DPBS, and the total RNA was collected using Isogen
II reagent (Nippon Gene, Tokyo, Japan). MDM control, M1 polarized, M1-M2 polarized, M2
polarized groups: 6 monocyte donors, 1 experiment, n = 6 per group; LP-PRP- and APS-addedgroups: 6 monocyte donors, 12 PRP donors, n = 72 per group.
4.7. Flow Cytometry
To collect cells, FACS buffer (DPBS + 1% BSA) was added to the Upcell Multi 24 wellplate (method 4.5.1). The plates were kept at room temperature for 30 min to promotedetachment of the cells and the cells were collected by pipetting the contents of each well.
FcR blocking reagent was added to the cell suspension and the cell suspension was kept atroom temperature for 15 min to inhibit Fc receptor-mediated nonspecific antibody binding.
Each cell suspension was divided into two tubes. In one tube, the cells were mixed withthe following six mouse monoclonal anti-human antibodies and the cell suspension was
kept at 4 ◦ C for 30 min for multiple staining: CD80-PE (Clone: L307.4), CD86-BUV395(Clone: 2331), CD163-FITC (Clone: GHI/61), CD206-BV421 (Clone: 19.2), CD14-APC(Clone: M5E2), CD45-BV605 (Clone: HI30) (BD Bioscience). Cells in the other tube weremixed with nonspecific fluorescent mouse IgGs as a negative control. The cells werereacted at 4 ◦ C for 30 min and then washed with FACS buffer. Data on the stained cells
were acquired using a BD LSR Fortessa ™ Flow Cytometer (BD Bioscience) and analyzed
using FlowJo (Tree Star, Ashland, OR, USA).
4.8. Gene Expression Analysis
Cells were lysed using Isogen II reagent for RNA extraction and stored at −80 ◦ C
until use. The lysate was then thawed at room temperature, and 40% (final volume)
deionized water was added. The sample was vortexed and centrifuged at 16,139× g and4 ◦ C for 15 min, 75% of the supernatant was transferred to a new tube, and an equal

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amount of 70% ethanol was added and then vortexed. Total RNA was extracted using an
RNeasy Micro kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol.
The total RNA quantity and quality were determined using a NanoDrop Lite (Thermo
Fisher Scientific). cDNA was synthesized from RNA (40 ng) using a QuantiTect reverse
transcription kit (QIAGEN) and a Thermal Cycler GeneAmp PCR System 9700 (Thermo
Fisher Scientific). cDNA (5 ng) was pre-amplified using TaqMan PreAmp Master Mix(Applied Biosystems, Waltham, MA, USA). For pre-amplification, the reaction volume
was adjusted to 10 µL according to the manufacturer’s instructions. Using the Thermal
Cycler GeneAmp PCR System 9700, the DNA was denatured at 95 ◦ C for 10 min, andthen at 95 ◦ C for 15 s and 60 ◦ C for 4 min for 14 cycles. The amplified product was
diluted 20-fold with TE buffer (1 ×) and used for qRT-PCR. qRT-PCR was performed using
TaqMan fast advanced master mix (Applied Biosystems) with a QuantStudio 3 Real-Time
PCR System (Applied Biosystems) at 50 ◦ C for 2 min, 95 ◦ C for 2 min, and then 95 ◦ C
for 1 s and 60 ◦ C for 20 s for 40 cycles. The probes used for pre-amplification and qRTPCR were GAPDH (Hs_02758991_g1), IL-1β (Hs_01555410_m1), IL-6 (Hs_00985639_m1),
TNF-α (Hs_00174128_m1), MRC1 (Hs_00267207_m1), IL-10 (Hs_00961622_m1), and TGF-β(Hs_00998133_m1). The value of each gene expression (-∆∆Ct value) was obtained against
the Ct of the internal control GAPDH and normalized to the control sample.
4.9. Statistical Analysis
Numerical results were statistically analyzed using SPSS® Statistics Software 26 (IBM,Armonk, NY, USA). The data obtained was tested for normality of distribution throughthe Kolomogorov–Smirnov test and was rejected. Thus, the Wilcoxon signed-rank test wasused for two-group comparisons, and Friedman’s test was used to compare three or moregroups. The significance level was set at p < 0.05.
5. Conclusions
PRPs purified using two clinically relevant kits contained different concentrations ofhumoral factors, among which both M1 and M2 macrophage-related factors were contained
at a higher concentration in APS. The addition of PRP supernatants to the culture media ofmonocytes and M1 macrophages promoted their polarization to M2 macrophages, possibly
in different ways. These results warrant a further investigation of the possibility that PRPs
may act on M1 macrophages in the joint and synovium to improve OAK symptoms.
Author Contributions: Conceptualization, M.S., E.T., and R.U.; data curation R.U. and M.M.; formal
analysis, R.U., investigation, R.U., M.M. and S.W.; resources, S.W., H.O. and M.W.; writing—originaldraft preparation, R.U.; writing—review and editing, M.S.; supervision, M.S. and M.W. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This study was reviewed and approved by the Institutional
Review Board of the Tokai University School of Medicine (18R-134) and was conducted in compliancewith relevant guidelines.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available on request from thecorresponding author. The data are not publicly available because of confidentiality issues.
Acknowledgments: We would like to express our deep gratitude to the Support Center for Medical
Research and Education for their cooperation in advancing this research.
Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2021, 22, 2336

14 of 16AbbreviationsAPS
CX3CL1CXCL9GAPDHGM-CSF
IFN-γ
IL-10
IL-13
IL-1β
IL-1RA
IL-4
IL-6
IP-10
LP-PRPLR-PRP
MCP-1
M-CSF
MDM
MRC1
OAK
PRP
TGF-β
TNF-α

Autologous protein solution
C-XXX-C motif chemokine ligand 1
C-X-C motif chemokine ligand 9
Glyceraldehyde 3-phosphate dehydrogenase
Granulocyte macrophage colony-stimulating factor
Interferon-gamma
Interleukin-10
Interleukin-13
Interleukin-1 beta
Interleukin-1 receptor antagonist
Interleukin-4
Interleukin-6
Interferon gamma-induced protein-10
Leukocyte-poor platelet rich plasma
Leukocyte-rich platelet rich plasma
Macrophage chemoattractant protein-1
Macrophage colony-stimulating factor
Monocyte derived macrophage
Mannose receptor 1
Osteoarthritis of the knee
Platelet rich plasma
Transforming growth factor beta
Tumor necrosis factor-alphaReferences
1.
2.
3.

4.

5.

6.
7.
8.

9.

10.
11.
12.

13.

Hügle, T.; Geurts, J. What drives osteoarthritis?—Synovialversussubchondral bone pathology. Rheumatology 2016, 56, 1461–1471.
[CrossRef]
Loeser, R.F.; Goldring, S.R.; Scanzello, C.R.; Goldring, M.B. Osteoarthritis: A disease of the joint as an organ. Arthritis Rheum.
2012, 64, 1697–1707. [CrossRef] [PubMed]
Yoshimura, N.; Muraki, S.; Oka, H.; Mabuchi, A.; En-Yo, Y.; Yoshida, M.; Saika, A.; Yoshida, H.; Suzuki, T.; Yamamoto, S.; et al.
Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: The research on osteoarthritis/osteoporosis against disability study. J. Bone Miner. Metab. 2009, 27, 620–628. [CrossRef] [PubMed]
McAlindon, T.E.; Bannuru, R.R.; Sullivan, M.C.; Arden, N.K.; Berenbaum, F.; Bierma-Zeinstra, S.M.; Hawker, G.A.; Henrotin, Y.;
Hunter, D.J.; Kawaguchi, H.; et al. Response to Letter to the Editor entitled “Comments on ‘OARSI guidelines for the non-surgicalmanagement of knee osteoarthritis’”. Osteoarthr. Cartil. 2014, 22, 890. [CrossRef] [PubMed]
Zhang, W.; Moskowitz, R.; Nuki, G.; Abramson, S.; Altman, R.; Arden, N.; Bierma-Zeinstra, S.; Brandt, K.; Croft, P.;
Doherty, M.; et al. OARSI recommendations for the management of hip and knee osteoarthritis, Part II: OARSI evidence-based,
expert consensus guidelines. Osteoarthr. Cartil. 2008, 16, 137–162. [CrossRef]
Woodell-May, J.; Matuska, A.; Oyster, M.; Welch, Z.; O’Shaughnessey, K.; Hoeppner, J. Autologous protein solution inhibits
MMP-13 production by IL-1β and TNFα-stimulated human articular chondrocytes. J. Orthop. Res. 2011, 29, 1320–1326. [CrossRef]
El-Sharkawy, H.; Kantarci, A.; Deady, J.; Hasturk, H.; Liu, H.; Alshahat, M.; Van Dyke, T.E. Platelet-Rich Plasma: Growth Factors
and Pro- and Anti-Inflammatory Properties. J. Periodontol. 2007, 78, 661–669. [CrossRef]
Kon, E.; Engebretsen, L.; Verdonk, P.; Nehrer, S.; Filardo, G. Clinical Outcomes of Knee Osteoarthritis Treated with an Autologous
Protein Solution Injection: A 1-Year Pilot Double-Blinded Randomized Controlled Trial. Am. J. Sports Med. 2018, 46, 171–180.
[CrossRef] [PubMed]Mourão, C.F.D.A.B.; De Mello-Machado, R.C.; Javid, K.; Moraschini, V. The use of leukocyte- and platelet-rich fibrin in the
management of soft tissue healing and pain in post-extraction sockets: A randomized clinical trial. J. Cranio-Maxillofac. Surg.
2020, 48, 452–457. [CrossRef] [PubMed]
Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 2003, 3, 23–35. [CrossRef] [PubMed]
Arnold, C.E.; Gordon, P.; Barker, R.N.; Wilson, H.M. The activation status of human macrophages presenting antigen determines
the efficiency of Th17 responses. Immunobiology 2015, 220, 10–19. [CrossRef] [PubMed]
Utomo, L.; Bastiaansen-Jenniskens, Y.; Verhaar, J.; Van Osch, G. Cartilage inflammation and degeneration is enhanced by proinflammatory (M1) macrophages in vitro, but not inhibited directly by anti-inflammatory (M2) macrophages. Osteoarthr. Cartil.
2016, 24, 2162–2170. [CrossRef] [PubMed]
Lee, H.-W.; Choi, H.-J.; Ha, S.-J.; Lee, K.-T.; Kwon, Y.-G. Recruitment of monocytes/macrophages in different tumor microenvironments. Biochim. et Biophys. Acta (BBA) Bioenerg. 2013, 1835, 170–179. [CrossRef]

Int. J. Mol. Sci. 2021, 22, 2336

14.
15.
16.
17.

18.

19.
20.
21.

22.
23.

24.
25.
26.
27.

28.
29.
30.
31.
32.
33.
34.
35.

36.

37.

38.
39.
40.

15 of 16

Mantovani, A.; Sica, A.; Sozzani, S.; Allavena, P.; Vecchi, A.; Locati, M. The chemokine system in diverse forms of macrophageactivation and polarization. Trends Immunol. 2004, 25, 677–686. [CrossRef] [PubMed]
Zhang, S.; Chuah, S.J.; Lai, R.C.; Hui, J.H.P.; Lim, S.K.; Toh, W. MSC exosomes mediate cartilage repair by enhancing proliferation,
attenuating apoptosis and modulating immune reactivity. Biomaterials 2018, 156, 16–27. [CrossRef]
Liu, B.; Zhang, M.; Zhao, J.; Zheng, M.; Yang, H. Imbalance of M1/M2 macrophages is linked to severity level of knee osteoarthritis.
Exp. Ther. Med. 2018, 16, 5009–5014. [CrossRef] [PubMed]
Sun, A.R.; Panchal, S.K.; Friis, T.; Sekar, S.; Crawford, R.; Brown, L.; Xiao, Y.; Prasadam, I. Obesity-associated metabolic syndrome
spontaneously induces infiltration of pro-inflammatory macrophage in synovium and promotes osteoarthritis. PLoS ONE 2017,12, e0183693. [CrossRef]
Manferdini, C.; Paolella, F.; Gabusi, E.; Gambari, L.; Piacentini, A.; Filardo, G.; Fleury-Cappellesso, S.; Barbero, A.; Murphy, M.;
Lisignoli, G. Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by
PGE2: In vitro evaluation. Osteoarthr. Cartil. 2017, 25, 1161–1171. [CrossRef]
Woodell-May, J.E.W.; Sommerfeld, S.D. Role of Inflammation and the Immune System in the Progression of Osteoarthritis.
J. Orthop. Res. 2020, 38, 253–257. [CrossRef] [PubMed]
Nishio, H.; Saita, Y.; Kobayashi, Y.; Takaku, T.; Fukusato, S.; Uchino, S.; Wakayama, T.; Ikeda, H.; Kaneko, K. Platelet-rich plasma
promotes recruitment of macrophages in the process of tendon healing. Regen. Ther. 2020, 14, 262–270. [CrossRef] [PubMed]
Omar, N.N.; Rashed, R.R.; El-Hazek, R.M.; El-Sabbagh, W.A.; Rashed, E.R.; El-Ghazaly, M.A. Platelet-rich plasma-induced
feedback inhibition of activin A/follistatin signaling: A mechanism for tumor-low risk skin rejuvenation in irradiated rats.
J. Photochem. Photobiol. B: Biol. 2018, 180, 17–24. [CrossRef]
Escobar, G.; Escobar, A.; Ascui, G.; Tempio, F.I.; Ortiz, M.C.; Pérez, C.A.; López, M.N. Pure platelet-rich plasma and supernatantof calcium-activated P-PRP induce different phenotypes of human macrophages. Regen. Med. 2018, 13, 427–441. [CrossRef]
Kon, E.; Di Matteo, B.; Delgado, D.; Cole, B.J.; Dorotei, A.; Dragoo, J.L.; Filardo, G.; Fortier, L.A.; Giuffrida, A.; Jo, C.H.; et al.
Platelet-rich plasma for the treatment of knee osteoarthritis: An expert opinion and proposal for a novel classification and coding
system. Expert Opin. Biol. Ther. 2020, 20, 1447–1460. [CrossRef] [PubMed]
Stout, R.D.; Jiang, C.; Matta, B.; Tietzel, I.; Watkins, S.K.; Suttles, J. Macrophages Sequentially Change Their Functional Phenotype
in Response to Changes in Microenvironmental Influences. J. Immunol. 2005, 175, 342–349. [CrossRef]
Gawaz, M.P.; Vogel, S. Platelets in tissue repair: Control of apoptosis and interactions with regenerative cells. Blood 2013, 122,
2550–2554. [CrossRef] [PubMed]
Knop, E.; De Paula, L.E.; Fuller, R. Platelet-rich plasma for osteoarthritis treatment. Rev. Bras. de Reum. (Engl. Ed.) 2016, 56,
152–164. [CrossRef]
Wasai, S.; Sato, M.; Maehara, M.; Toyoda, E.; Uchiyama, R.; Takahashi, T.; Okada, E.; Iwasaki, Y.; Suzuki, S.; Watanabe, M.
Characteristics of autologous protein solution and leucocyte-poor platelet-rich plasma for the treatment of osteoarthritis of theknee. Sci. Rep. 2020, 10, 1–11. [CrossRef] [PubMed]
Mussano, F.; Genova, T.; Munaron, L.; Petrillo, S.; Erovigni, F.; Carossa, S. Cytokine, chemokine, and growth factor profile ofplatelet-rich plasma. Platelets 2016, 27, 467–471. [CrossRef]
Castillo, T.N.; Pouliot, M.A.; Kim, H.J.; Dragoo, J.L. Comparison of Growth Factor and Platelet Concentration From Commercial
Platelet-Rich Plasma Separation Systems. Am. J. Sports Med. 2010, 39, 266–271. [CrossRef]
Julier, Z.; Park, A.J.; Briquez, P.S.; Martino, M.M. Promoting tissue regeneration by modulating the immune system. Acta Biomater.
2017, 53, 13–28. [CrossRef]
Novak, M.L.; Koh, T.J. Macrophage phenotypes during tissue repair. J. Leukoc. Biol. 2013, 93, 875–881. [CrossRef] [PubMed]
Benoit, M.; Desnues, B.; Mege, J.-L. Macrophage Polarization in Bacterial Infections. J. Immunol. 2008, 181, 3733–3739. [CrossRef]
Gordon, S.; Taylor, P.R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 2005, 5, 953–964. [CrossRef] [PubMed]
Mantovani, A.; Biswas, S.K.; Galdiero, M.R.; Sica, A.; Locati, M. Macrophage plasticity and polarization in tissue repair andremodelling. J. Pathol. 2013, 229, 176–185. [CrossRef] [PubMed]
Pessler, F.; Dai, L.; Diaz-Torne, C.; Gomez-Vaquero, C.; Paessler, M.E.; Zheng, D.-H.; Einhorn, E.; Range, U.; Scanzello, C.;
Schumacher, H.R. The synovitis of “non-inflammatory” orthopaedic arthropathies: A quantitative histological and immunohistochemical analysis. Ann. Rheum. Dis. 2008, 67, 1184–1187. [CrossRef] [PubMed]
Zhang, H.; Lin, C.; Zeng, C.; Wang, Z.; Wang, H.; Lu, J.; Liu, X.; Shao, Y.; Zhao, C.; Pan, J.; et al. Synovial macrophage M1
polarisation exacerbates experimental osteoarthritis partially through R-spondin-2. Ann. Rheum. Dis. 2018, 77, 1524–1534.
[CrossRef] [PubMed]
O’Shaughnessey, K.; Matuska, A.; Hoeppner, J.; Farr, J.; Klaassen, M.; Kaeding, C.; Lattermann, C.; King, W.; Woodell-May, J.
Autologous protein solution prepared from the blood of osteoarthritic patients contains an enhanced profile of anti-inflammatory
cytokines and anabolic growth factors. J. Orthop. Res. 2014, 32, 1349–1355. [CrossRef]
Wojdasiewicz, P.; Poniatowski, Ł.A.; Szukiewicz, D. The Role of Inflammatory and Anti-Inflammatory Cytokines in the Pathogenesis of Osteoarthritis. Mediat. Inflamm. 2014, 2014, 1–19. [CrossRef]Rőszer, T. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms. Mediat. Inflamm.
2015, 2015, 1–16. [CrossRef]
Vogel, D.Y.; Glim, J.E.; Stavenuiter, A.W.; Breur, M.; Heijnen, P.; Amor, S.; Dijkstra, C.D.; Beelen, R.H. Human macrophage
polarization in vitro: Maturation and activation methods compared. Immunobiology 2014, 219, 695–703. [CrossRef]

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42.

16 of 16

Bertani, F.R.; Mozetic, P.; Fioramonti, M.; Iuliani, M.; Ribelli, G.; Pantano, F.; Santini, D.; Tonini, G.; Trombetta, M.;
Businaro, L.; et al. Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal
microscopy and multivariate analysis. Sci. Rep. 2017, 7, 1–9. [CrossRef] [PubMed]
Tjiu, J.-W.; Chen, J.-S.; Shun, C.-T.; Lin, S.-J.; Liao, Y.-H.; Chu, C.-Y.; Tsai, T.-F.; Chiu, H.-C.; Dai, Y.-S.; Inoue, H.; et al. TumorAssociated Macrophage-Induced Invasion and Angiogenesis of Human Basal Cell Carcinoma Cells by Cyclooxygenase-2
Induction. J. Investig. Dermatol. 2009, 129, 1016–1025. [CrossRef] [PubMed]