Winner of the 2016 Cabaud Award

Platelet-Rich Plasma Activates

Proinflammatory Signaling Pathways
and Induces Oxidative Stress
in Tendon Fibroblasts
Joshua L. Hudgens,* MD, Kristoffer B. Sugg,*yz MD, Jeremy A. Grekin,* MS,
Jonathan P. Gumucio,*y BS, Asheesh Bedi,* MD, and Christopher L. Mendias,*y§ PhD, ATC
Investigation performed at the Department of Orthopaedic Surgery,
University of Michigan Medical School, Ann Arbor, Michigan, USA

Background: Tendon injuries are one of the most common musculoskeletal conditions in active patients. Platelet-rich plasma
(PRP) has shown some promise in the treatment of tendon disorders, but little is known as to the mechanisms by which PRP
can improve tendon regeneration. PRP contains numerous different growth factors and cytokines that activate various cellular
signaling cascades, but it has been difficult to determine precisely which signaling pathways and cellular responses are activated
after PRP treatment. Additionally, macrophages play an important role in modulating tendon regeneration, but the influence of
PRP on determining whether macrophages assume a proinflammatory or anti-inflammatory phenotype remains unknown.
Purpose: To use genome-wide expression profiling, bioinformatics, and protein analysis to determine the cellular pathways acti-
vated in fibroblasts treated with PRP. The effect of PRP on macrophage polarization was also evaluated.
Study Design: Controlled laboratory study.
Methods: Tendon fibroblasts or macrophages from rats were cultured and treated with either platelet-poor plasma (PPP) or PRP.
RNA or protein was isolated from cells and analyzed using microarrays, quantitative polymerase chain reaction, immunoblotting,
or bioinformatics techniques.
Results: Pathway analysis determined that the most highly induced signaling pathways in PRP-treated tendon fibroblasts were
TNFa and NFkB pathways. PRP also downregulated the expression of extracellular matrix genes and induced the expression of
autophagy-related genes and reactive oxygen species (ROS) genes and protein markers in tendon fibroblasts. PRP failed to have
a major effect on markers of macrophage polarization.
Conclusion: PRP induces an inflammatory response in tendon fibroblasts, which leads to the formation of ROS and the activation
of oxidative stress pathways. PRP does not appear to significantly modulate macrophage polarization.
Clinical Relevance: PRP might act by inducing a transient inflammatory event, which could then trigger a tissue regeneration
response.
Keywords: platelet-rich plasma; tendon; tendinopathy; oxidative stress; inflammation; autophagy

Acute and chronic tendon injuries are relatively common centrifugation.1 Platelets are important in the injury
problems in the general population, secondary to sports response, as they release growth factors that initiate and
participation and other physical activity, and may be the modulate wound healing in both soft and hard tissues.
source of significant morbidity.8,24 Platelet-rich plasma The justification for the use of PRP clinically stems from
(PRP) is a commonly used biological treatment in sports an attempt to recapitulate or augment this natural biolog-
medicine, specifically with interstitial tendon tears and ical process.1 Despite numerous clinical outcome studies on
chronic tendinopathies.8,25 Initially described for use in the effects of PRP in sports medicine, there remains a pau-
oral and maxillofacial surgery, PRP is a refined product city of information on its mechanism of action.19,25
of autologous blood with a platelet concentration greater Two cell types, fibroblasts and macrophages, appear to
than that of whole blood, typically isolated via differential predominate and coordinate the healing process in injured
and diseased tendons.9,37,39 Fibroblasts function as the
principal cells involved in tendon maintenance and repair,
while macrophages help to break down damaged tendon
The American Journal of Sports Medicine, Vol. 44, No. 8
DOI: 10.1177/0363546516637176 tissue and can secrete cytokines and other signaling mole-
Ó 2016 The Author(s) cules that modulate the activity of fibroblasts.26,28,38,39

1931
1932 Hudgens et al The American Journal of Sports Medicine

Macrophages exhibit 2 phenotypes: a proinflammatory rats, a manual preparation approach modified from previ-
(M1) phenotype and an anti-inflammatory (M2) pheno- ous studies1,10 was used. Briefly, blood was centrifuged at
type.28,38 In response of tissues to injury, the M1 popula- 500g for 5 minutes at 4°C, followed by a 5-minute rest
tion of macrophages predominates initially, mediating period, and then another cycle again at 700g for
phagocytosis and apoptosis, while M2 macrophages appear 17 minutes at 4°C. The supernatant above the packed cells
later and become the more prevalent population that coor- contained 2 visibly different layers: the uppermost and
dinates the repair process and promotes fibroblast prolifer- nearly transparent layer containing PPP and a lower par-
ation.7,28,39 There have been several in vitro studies on the tially flocculent layer containing PRP. A total of 3 separate
effect of PRP on tendon cells, measuring the expression of batches of PRP and PPP were prepared. A Hemavet 950
specific genes or proteins related to tendon function,40,41 system (Drew Scientific) was used to quantify platelet den-
and to our knowledge, no studies of the effect of PRP on sities. The mean (6SD) concentration of platelets from
macrophage polarization. As PRP is a dense milieu of PRP was 1.4 3 106 6 0.5 3 105 platelets/mL and from
numerous growth factors and other signaling molecules, PPP was 3.0 3 104 6 0.5 3 103 platelets/mL, which
and only a limited number of signaling pathways have resulted in PRP having an approximately 4-fold elevation
been studied in tendon cells treated with PRP, we sought in platelet concentration compared with whole blood.
to determine transcriptome-wide changes in gene expres- PRP and PPP were frozen at 280°C until use.
sion using microarrays and informative bioinformatics
analyses to evaluate which cellular signaling pathways
were activated by PRP in an unbiased fashion. Further- Tendon Fibroblast Culture
more, because macrophages appear to play an important
role in tendon inflammation and repair, we determined Fibroblasts were isolated and from tail tendons as previ-
the effect of PRP on macrophage polarization. We hypoth- ously described.31 Tail tendons develop from the same pop-
esized that PRP would activate signaling pathways ulation of somitic progenitor cells as limb tendons33 and
involved in extracellular matrix (ECM) synthesis and are useful in obtaining a large number of low passage cells.
remodeling and would polarize macrophages to an anti- Tendon fascicles were finely minced and placed in Dulbec-
inflammatory M2 phenotype. co’s Modified Eagle Medium (DMEM) containing 0.2% type
2 collagenase (Life Technologies) and vigorously agitated
for 3 hours at 37°C. An equal volume of growth medium
METHODS (GM) composed of DMEM containing 10% fetal bovine
serum (FBS) and 1% antibiotic/antimycotic (Life Technolo-
Animals gies) was added to the digested tissue, which was then fil-
tered through a 100-mm strainer. Cells were centrifuged at
This study was approved by the University of Michigan 1000g for 10 minutes, resuspended in GM, and plated on
Institutional Animal Care and Use Committee and fol- 100-mm type 1 collagen–coated dishes (BD). All cells
lowed United States Public Health Service guidelines for were cultured in humidified incubators maintained at
the ethical treatment of animals. Male retired-breeder 37°C and 5% CO2. Fibroblasts were grown to 70% conflu-
inbred Lewis rats were obtained from Charles River Labo- ence, collected from dishes using TrypLE (Life Technolo-
ratories and were housed under specific pathogen-free con- gies), and resuspended in 3-dimensional (3D) type 1
ditions. The inbred Lewis strain was selected to avoid collagen gels. The collagen for these gels was prepared
adverse immune reactions from blood pooling.29 Rats and extracted as described previously.18 Briefly, rat tail
were deeply anesthetized with sodium pentobarbital to tendons were excised and placed in 0.2% acetic acid at
obtain blood and tendon tissue and then humanely eutha- 4°C. After 5 days, the collagen solution was centrifuged
nized via an anesthetic overdose and the induction of at 24,000g for 30 minutes, and the supernatant was col-
a bilateral pneumothorax. lected, lyophilized, and dissolved again in 0.2% acetic
acid to a final concentration of 2.7 mg/mL. To prepare
Plasma Preparation the collagen gel, the collagen solution was combined with
103 minimum essential medium (Life Technologies) and
Blood was obtained via cardiac puncture and collected into 0.34 N NaOH in an 8:1:1 ratio at 4°C. Tendon fibroblasts
sodium citrate Vacutainer tubes (BD). Platelet-poor were resuspended in this mixture, and 300 mL containing
plasma (PPP) and PRP were prepared from whole blood 2 3 105 cells was added to each well of a 24-well plate
under sterile conditions. As no widely used commercially (BD). The plate was then placed in the humidified incuba-
available clinical system is available to prepare PRP from tor at 37°C for 45 minutes for gelling to occur.

§
Address correspondence to Christopher L. Mendias, PhD, ATC, Department of Orthopaedic Surgery, University of Michigan Medical School, 109 Zina
Pitcher Place, BSRB 2017, Ann Arbor, MI 48109-2200, USA (email: [email protected]).
*Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.
y
Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
z
Section of Plastic Surgery, Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Presented at the annual meeting of the AOSSM, Colorado Springs, Colorado, July 2016.
One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by National Institutes
of Health grants R01-AR063649, F31-AR065931, and F32-AR067086.
AJSM Vol. 44, No. 8, 2016 PRP Induces Oxidative Stress in Tendon Fibroblasts 1933

After being embedded in 3D collagen gels, fibroblasts were (ascension No. GSE70918). The Upstream Regulator mod-
cultured in GM for 3 days. The medium was then changed to ule of Ingenuity Pathway Analysis (IPA) software (Qiagen)
contain DMEM plus 1% antibiotic/antimycotic and 10% PPP was used to determine the transcriptional regulators that
or PRP clot releasate. The clot releasate was prepared by could explain the observed change in gene expression
treating PPP or PRP with 30 mM CaCl2 to activate the coag- measurements obtained from microarrays. This module
ulation cascade. Activated PPP and PRP were vigorously agi- examines the number of known targets of each transcrip-
tated for 1 hour at 4°C and then spun at 12,000g for tion regulator that are present in the microarray dataset,
10 minutes at 4°C. The supernatant containing the clot relea- along with the direction of change to predict likely relevant
sate was collected, added to DMEM plus 1% antibiotic/ transcriptional regulators. If the observed direction of the
antimycotic, and passed through a 0.22-mm filter to remove fold change in expression is consistent with a particular
any small fibrin clumps. The resulting PPP- or PRP-contain- activation state of the transcriptional regulator, then a pre-
ing medium was added to wells containing tendon fibroblasts diction is made about whether the pathway is activated or
and changed every 2 days. inhibited. A full listing of the IPA Upstream Regulator
results is listed in Appendix Table A2 (available online).
Macrophage Culture
Rat resident peritoneal macrophages were purchased from Protein Isolation and Measurements
Cell Biologics and were cultured in macrophage medium con-
Fibroblasts in 3D collagen gels were treated with media
taining basal medium supplemented with granulocyte mac-
containing PPP or PRP for 5 days. Collagen gels were
rophage colony-stimulating factor, 10% FBS, and 1%
homogenized in ice-cold radioimmunoprecipitation assay
antibiotic/antimycotic (Cell Biologics). Cells were thawed
buffer (Sigma-Aldrich) supplemented with 1:100 protease
and cultured on plasma-treated dishes (BD) for 3 days, after
and phosphatase inhibitor cocktail (Life Technologies)
which 10% FBS in the medium was substituted for either
and 1% NP-40 (Sigma-Aldrich). After vigorous homogeni-
10% PPP or PRP for 2 days before RNA isolation.
zation, samples were vortexed for 10 minutes at 4°C and
then spun at 12,000g for 10 minutes at 4°C. The superna-
RNA Isolation and Gene Expression tant was collected, and the protein concentration was mea-
sured using a bicinchoninic acid assay (Life Technologies).
Tendon fibroblasts or macrophages were treated with PPP
Proteins were diluted in Laemmli sample buffer (Bio-Rad),
or PRP for 24 hours, and RNA was isolated as previously
and 10 mg of total protein was loaded into Any kD gels (Bio-
described16,36 using an miRNeasy Micro Kit (Qiagen). All
Rad). Proteins were separated with electrophoresis, and
RNA had an A260/A280 ratio .1.8 (NanoDrop; Thermo
the gels were either stained with Coomassie Brilliant
Fisher) and RNA integrity number (RIN) values .8.0 mea-
Blue (Bio-Rad) or transferred to membranes for immuno-
sured (Bioanalyzer; Agilent). After reverse transcription of
blotting (Bio-Rad). For NFkB immunoblots, nitrocellulose
RNA with iScript Supermix (Bio-Rad), quantitative poly-
membranes were blocked in 2% goat serum and incubated
merase chain reaction (qPCR) was conducted in a CFX96
with rabbit anti–phospho-NFkB antibodies (S536, 1:1000
real-time thermal cycler using iTaq SYBR green supermix
dilution) and goat anti-rabbit horseradish peroxidase
reagents (Bio-Rad). The 2–DCt technique was used to nor-
(HRPO)–conjugated secondary antibodies (1:2000 dilu-
malize the expression of mRNA transcripts to the stable
tion). For the detection of carbonylated proteins, an OxiSe-
housekeeping gene b-actin. A listing of RNA transcripts
lect Protein Carbonyl Immunoblot Kit (Cell Biolabs) was
and primer sequences is provided in Appendix Table A1
used following manufacturer directions. Briefly, polyviny-
(available online at http://ajsm.sagepub.com/supplemental).
lidene difluoride membranes were blocked in 5% powdered
milk and treated with dinitrophenylhydrazine, which
Microarray Analysis reacts with carbonylated amino acid residues in proteins
to produce dinitrophenol residues. Membranes were then
Microarray measurements of PPP- or PRP-treated tendon
incubated with anti-dinitrophenol residue antibodies
fibroblasts were performed by the University of Michigan
(1:1000 dilution) and HRPO-conjugated secondary antibod-
DNA Sequencing Core following manufacturer recommen-
ies (1:1000 dilution). Membranes were treated with
dations. Equal amounts of RNA isolated from 3 individual
enhanced chemiluminescence solution (Clarity ECL; Bio-
wells were pooled into a single sample for microarray anal-
Rad) to activate HRPO. Gels and membranes were visual-
ysis, and 2 pooled samples of PPP and 2 pooled samples of
ized in a ChemiDoc XRS system (Bio-Rad), and densitome-
PRP were analyzed. RNA was pooled because gene expres-
try analysis was performed using Image Lab 5.2 software
sion from a pooled RNA sample is similar to the average
(Bio-Rad).
from the individual samples composing the pooled sam-
ple.5,23 RNA was prepared for microarray analysis using
a GeneChip WT Pico Kit (Affymetrix) and hybridized to Protein Array
Rat Gene ST 2.1 strips (Affymetrix). Raw microarray
data were loaded into ArrayStar version 12.1 (DNASTAR) A rat cytokine antibody array (C2; RayBiotech) was used to
to calculate fold changes in gene expression data. The measure the abundance of 34 proteins in PPP and PRP sam-
microarray dataset is available through the National Insti- ples. A total of 100 mL of PPP or PRP was used per assay, which
tutes of Health Gene Expression Omnibus database followed the instructions of the manufacturer. Membranes were
1934 Hudgens et al The American Journal of Sports Medicine

developed with the Clarity ECL solution and quantified as TABLE 1

described above. Abbreviated Terms

Abbreviation Expansion
Statistical Analysis
Arg Arginase
Data are presented as mean 6 SD. Differences between Atg Autophagy-related protein
PPP and PRP groups were assessed using t tests (a = .05) BMP Bone morphogenetic protein
in GraphPad Prism 6.0. Bnip B-cell lymphoma/adenovirus E1B
interacting protein
CCL Chemokine (C-C motif) ligand
RESULTS CCR C-C chemokine receptor
CD Cluster of differentiation
Protein Abundance in PPP Versus PRP CILP Cartilage intermediate layer protein
CNTF Ciliary neurotrophic factor
The relative difference in cytokines, growth factors, and Col Collagen
Cox Cyclooxygenase
other proteins important in tissue inflammation and macro-
CSF Colony-stimulating factor
phage activity in PPP and PRP was determined. Abbreviated CTGF Connective tissue growth factor
terms used in this article are shown in Table 1. Of the 34 pro- CXCL Chemokine (C-X-C motif) ligand
teins analyzed, 26 were significantly higher in PRP com- EGR Early growth response protein
pared with PPP, including CCL2, CCL20, CXCL5, IL1a, FACIT Fibril-associated collagens with interrupted
IL1b, IL6, IL10, PDGF-AA, and TNFa (Table 2). triple helices
FGF Fibroblast growth factor
Fmod Fibromodulin
Microarray and Bioinformatics GABARAPL Gamma-aminobutyric acid receptor-associated
Analysis of PRP Treatment protein–like
HAS Hyaluronan synthase
We analyzed the effect of PRP treatment on global changes IFN Interferon
in gene expression patterns in tendon fibroblasts. Using IGF Insulin-like growth factor
microarrays, we determined that PRP treatment resulted IKK IkB kinase
in an upregulation greater than 1.5-fold of 315 genes and IL Interleukin
a downregulation greater than 1.5-fold of 460 genes (Figure iNOS Inducible nitric oxide synthase
1A). To gain more information about the biological signifi- LOX Lipoxygenase
cance of the microarray results and identify the signaling MMP Matrix metalloproteinase
pathways predicted to be activated or inhibited by PRP, NFE2L Nuclear factor (erythroid-derived 2)–like
NFkB Nuclear factor kappa light chain enhancer of
we analyzed fold changes in global gene expression with
activated B cells
the Upstream Regulator module of the IPA software. This PDGF Platelet-derived growth factor
analysis identified the TNFa pathway (P = 6.6 3 1026) PLD1 Phospholipase D1
and the NFkB pathway (P = 9.5 3 10219) as the 2 pathways Prdx Peroxiredoxin
activated in fibroblasts treated with PRP (Figure 1B). PTGES Prostaglandin E synthase
Scx Scleraxis
SIRT Sirtuin
PRP Effects on Tendon Fibroblast Gene Expression
SOD Superoxide dismutase
TGF Transforming growth factor
After performing microarrays, we sought to validate the
TIMP Tissue inhibitor of metalloproteinases
fold change values of specific genes of relevance to tendon
TNFa Tumor necrosis factor alpha
biology and inflammation with qPCR. For genes related to TNFR Tumor necrosis factor receptor
tendon growth, PRP upregulated BMP7 but did not change Tnmd Tenomodulin
the expression of TGFb and downregulated CTGF and Trim Tripartite motif containing
IGF1 (Figure 2). The inflammation- and immune-modulat- VEGF Vascular endothelial growth factor
ing cytokines CCL2, CCL7, IL1a, IL6, IL10, and TNFa
were upregulated in response to PRP, while no difference
in IL1b or VEGF was observed, and IL15 was downregu- PRP-treated fibroblasts, while the basement membrane
lated (Figure 2). type 8 collagen and the proteoglycan lubricin were upregu-
The expression of genes involved with ECM synthesis lated (Figure 3). PRP induced the expression of the major
and remodeling was also quantified. PRP had no effect on collagenase MMP13, along with the stromelysins MMP3
the expression of the hyaluronic acid (HA) synthase and MMP10 and the gelatinase MMP9, with no difference
enzymes HAS1 and HAS2 (Figure 3). Elastin expression observed in the expression of the collagenase MMP8 and
was downregulated along with a slight elevation in the the gelatinase MMP2 nor the TIMP genes TIMP1 or
cross-linking enzyme LOX (Figure 3). The major fibrillar TIMP2 (Figure 3).
collagens, type 1 and type 3 collagen, along with genes asso- Subsequently, the expression of genes involved in vari-
ciated with collagen fibril assembly, CILP, fibromodulin, ous cell functions including fibroblast proliferation, differ-
and collagen types 12 and 14, were downregulated in entiation, autophagy, and inflammation was assessed.
AJSM Vol. 44, No. 8, 2016 PRP Induces Oxidative Stress in Tendon Fibroblasts 1935

TABLE 2
Relative Abundance of 34 Different Proteins From Platelet-Poor Plasma (PPP) and Platelet-Rich Plasma (PRP)a

Protein PPP PRP

Activin A 1.00 6 0.51 2.07 6 0.21b

Advanced glycosylation end product (AGE) 1.00 6 0.56 2.09 6 0.22b
Agrin 1.00 6 0.32 1.95 6 0.04b
Brain-derived neurotrophic factor (BDNF) 1.00 6 0.54 2.01 6 0.12b
Chemokine (C-C motif) ligand 2 (CCL2) 1.00 6 0.48 2.87 6 0.08b
Chemokine (C-C motif) ligand 20 (CCL20) 1.00 6 0.30 2.04 6 0.11b
Chemokine (C-X-C motif) ligand 1 (CXCL1) 1.00 6 0.51 1.49 6 0.19
Chemokine (C-X-C motif) ligand 2 (CXCL2) 1.00 6 0.35 1.31 6 0.22
Chemokine (C-X-C motif) ligand 3 (CXCL3) 1.00 6 0.45 1.31 6 0.25
Chemokine (C-X-C motif) ligand 5 (CXCL5) 1.00 6 0.48 6.32 6 0.05b
Chemokine (C-X-C motif) ligand 7 (CXCL7) 1.00 6 0.29 1.36 6 0.11
Ciliary neurotrophic factor (CNTF) 1.00 6 0.53 2.00 6 0.26b
Cluster of differentiation 86 (CD86) 1.00 6 0.39 1.79 6 0.07b
Colony-stimulating factor 2 (CSF2) 1.00 6 0.52 2.29 6 0.06b
Fas ligand 1.00 6 0.79 2.83 6 0.36b
Fractalkine 1.00 6 0.57 3.04 6 0.17b
Intercellular adhesion molecule 1 (ICAM1) 1.00 6 0.49 2.74 6 0.01b
Interferon gamma (IFNg) 1.00 6 0.45 2.22 6 0.01b
Interleukin 1 receptor–like 2 (IL1RL2) 1.00 6 0.56 2.12 6 0.08b
Interleukin 1 alpha (IL1a) 1.00 6 0.32 1.90 6 0.05b
Interleukin 1 beta (IL1b) 1.00 6 0.43 2.17 6 0.07b
Interleukin 2 (IL2) 1.00 6 0.42 1.69 6 0.11
Interleukin 4 (IL4) 1.00 6 0.46 1.94 6 0.19b
Interleukin 6 (IL6) 1.00 6 0.40 2.01 6 0.17b
Interleukin 10 (IL10) 1.00 6 0.40 2.02 6 0.21b
Interleukin 13 (IL13) 1.00 6 0.56 1.45 6 0.32
L-selectin 1.00 6 0.46 2.83 6 0.05b
Leptin 1.00 6 0.46 2.43 6 0.03b
Matrix metalloproteinase 8 (MMP8) 1.00 6 0.27 4.17 6 0.01b
Platelet-derived growth factor AA (PDGF-AA) 1.00 6 0.32 1.73 6 0.01b
Prolactin receptor 1.00 6 0.50 1.53 6 0.05
Tissue inhibitor of metalloproteinases 1 (TIMP1) 1.00 6 0.40 3.01 6 0.26b
Tumor necrosis factor alpha (TNFa) 1.00 6 0.52 2.98 6 0.24b
Vascular endothelial growth factor A (VEGF-A) 1.00 6 0.52 1.57 6 0.01

a
Values are relative intensities normalized to the PPP group and are presented as mean 6 SD; n = 3 replicates from each group.
b
Significantly different from the PPP group (P \ .05).

The cell proliferation marker Ki67 was slightly elevated in PRP Effects on NFkB Activation
response to PRP treatment, but a downregulation in the and Protein Carbonylation
expression of genes involved in tendon fibroblast specifica-
tion and differentiation including EGR1, EGR2, scleraxis, To verify that an elevation in ROS was indeed present and
and tenomodulin was observed (Figure 4). For genes that the predicted elevation of NFkB did occur, we mea-
involved with autophagy, there was an induction in the sured the levels of carbonylated proteins and the abun-
expression of Atg10, Bnip1, and GABARAPL2, with no dif- dance of phospho-NFkB using immunoblots and observed
ference in beclin 1 or Trim13 levels (Figure 4). Transcrip- an induction in both the amount of carbonylated proteins
tion factors involved with inflammation, Fosb, Fosl1, and and in activated NFkB protein levels (Figure 5).
c-Jun, were induced by PRP, but no difference in the
expression of the deacetylase SIRT1 or the nitric oxide– PRP and Macrophage Polarization
producing gene iNOS was observed (Figure 4). Genes
involved with prostaglandin production were upregulated Finally, the expression of transcripts involved in the polar-
by PRP treatment, including PLD1, PTGES, Cox1, and ization of macrophages to a proinflammatory or anti-
Cox2, while no difference in the leukotriene synthesis inflammatory phenotype was assessed. PRP resulted in
enzyme 5-LOX was observed (Figure 4). PRP also induced an induction in the expression of the M1 proinflammatory
the expression of markers of elevated reactive oxygen spe- markers iNOS, IL1b, and VEGF, with no changes in the
cies (ROS) production, including SOD1, SOD2, NFE2L2, expression of CCR7, CD11b, CD68, IL15, or TNFa (Figure
and Prdx1 (Figure 4). 6A). However, there was also a modest induction in several
1936 Hudgens et al The American Journal of Sports Medicine

A 315 >1.5-fold  for PRP

P ≥ .05
33,389

3296 P < .05 2521

460 >1.5-fold  for PRP

B Upregulated
Downregulated
Predicted activation
Predicted inhibition
Leads to activation
Leads to inhibition

Figure 1. Microarray and bioinformatics analysis of tendon fibroblasts treated with platelet-rich plasma (PRP) or platelet-poor
plasma (PPP). (A) Microarray analysis identified 3296 genes of 36,685 that were significantly different (P \ .05) between PRP-
and PPP-treated cells. Of the 3296 genes that were significantly different, 315 genes were .1.5-fold upregulated, and 460 genes
were .1.5-fold downregulated in PRP-treated cells compared to PPP-treated cells. (B) Ingenuity Pathway Analysis identified 2
pathways that were predicted to be highly activated in cells treated with PRP compared to PPP: the TNFa pathway (P = 6.6 3
1026) and the NFkB pathway (P = 9.5 3 10219). The merged pathways are presented.

M2 anti-inflammatory markers including arginase 1, IL10, is the first study that investigated global transcriptomic
CD163, and CD14, with no change in FGF2, CD206, changes in tendon fibroblasts after PRP administration.
CD168, TGFb, or IGF1 expression (Figure 6B). We hypothesized that PRP would activate signaling path-
ways involved in ECM synthesis and remodeling. Surpris-
ingly, the only 2 pathways predicted to be activated were
DISCUSSION the proinflammatory TNFa and NFkB pathways. Genes
related to cellular proliferation and tendon collagen remod-
PRP is commonly used in the treatment of acute and chronic eling were also increased, suggesting that PRP may activate
tendon injuries and diseases,8,25 and to our knowledge, this fibroblast activity and collagen remodeling but not collagen
AJSM Vol. 44, No. 8, 2016 PRP Induces Oxidative Stress in Tendon Fibroblasts 1937

40
40 * PPP
PRP
*
10 * * * 10
* *

Relative Expression
Relative Expression

* *
* * *
*
1 1

*
* * * *

0.1 * 0.1
*
F
L2

L7
P7

F1

F
1b

10

15

F
F
TG

IL
1

G
C

TN
TG
IG

IL

IL

IL
IL
BM

13
x

1
1

sb
VE

67

L2
0
C

Sc
C

sl
in
R

ip
g1

im

Fo
Ki

AP

Fo
EG

EG

cl
Tn

Bn
At

Be

Tr

AR
AB
Figure 2. Gene expression of growth factor and cytokine tran-

G
scripts from tendon fibroblasts treated with platelet-rich plasma
(PRP) or platelet-poor plasma (PPP). Target gene expression 40 *
PPP
was normalized to b-actin. Values are mean 6 SD; n = 6 repli- PRP
cates from each group. *Significantly different from the PPP 10 * *
* *

Relative Expression
group (P \ .05).
* * * *
40
1

10 *
Relative Expression

* 0.1
*
S
n

T1

2
ES

1
x
1

2
Lo
Ju

ox

ox

dx
2L
D
O
R

SO

SO
PL

G
1
iN
c-

Pr
5-

FE
SI

PT

N
* * * Figure 4. Gene expression of cell proliferation, differentia-
0.1
* * tion, autophagy, and inflammatory transcripts from tendon
fibroblasts treated with platelet-rich plasma (PRP) or plate-
in
1

tin
1

let-poor plasma (PPP). Target gene expression was normal-

AS

AS

LO

ic
as
1

12

14

br
ol

ol

ol

El
ol

ol

Lu
C

ized to b-actin. Values are mean 6 SD; n = 6 replicates

C

from each group. *Significantly different from the PPP group

40
* * PPP (P \ .05).
PRP

10
treatment on macrophage polarization. While more studies
Relative Expression

* are necessary, these results provide important insight into

* the regulation of tendon cell activity by PRP treatment
and suggest that PRP might act by inducing an intermittent
1
bout of inflammation, which may then trigger a tissue
regeneration response.
PRP contains numerous growth factors and cytokines
0.1 that can activate various signaling pathways in cells.
* * Some of the signaling components that are downstream
P2

P3

P8

P9

P1

P2

od
P
P1

P1

of individual receptors can act to inhibit or further enhance

IL

Fm
M

C
M

M
M

TI

TI
M

the activation of other pathways regulated by different

receptors. For example, PRP contains both IL1b and
Figure 3. Gene expression of extracellular matrix structural
IL10. While IL1b is a well-known activator of proinflam-
and remodeling transcripts from tendon fibroblasts treated
matory intracellular signaling cascades, IL10 signaling
with platelet-rich plasma (PRP) or platelet-poor plasma
pathways are able to inhibit the pathways activated by
(PPP). Target gene expression was normalized to b-actin.
IL1b and reduce the expression of proinflammatory
Values are mean 6 SD; n = 6 replicates from each group.
genes.22 With the use of expression data from the entire
*Significantly different from the PPP group (P \ .05).
transcriptome, the IPA-based bioinformatics approach
employed in this study allowed us to evaluate the complex
synthesis. We also hypothesized that PRP would polarize relationship between various individual signaling cascades
macrophages to an anti-inflammatory M2 phenotype but to identify which pathways were most highly enriched
unexpectedly failed to observe any clear effect of PRP after PRP treatment. Interestingly, the highly related
1938 Hudgens et al The American Journal of Sports Medicine

A PPP PRP
B A
kDa
2.0
10 *

Carbonylation Rel Units

*

Relative Expression
1.5
Carbonylation

250 *
*
100 1.0
1
75
0.5
50

0.0
PPP PRP
pNFκB

C
75 0.1
10 *

S
68
b

F
0

15
50

F
11
/8

G
O
D

TN
IL
IL
C

VE
iN
F4

C
C
pNFκB Rel Units
8

C
250
6 B
Total Protein

10 PPP
100 4
PRP
75 2

Relative Expression
* *
50 0
* *
PPP PRP
1

Figure 5. Immunoblots for carbonylation and NFkB activa-

tion. (A) Immunoblots for total carbonylated proteins and
phospho-NFkB activation from tendon fibroblasts treated 0.1
with platelet-rich plasma (PRP) or platelet-poor plasma

F2

14

F1
g1

10

F
16

16

20
(PPP). A Coomassie-stained gel of the same samples is

D
Ar

FG

TG
IG

IL
D

D
C

C
shown for validation of equal protein loading. Band densi-
tometry analysis for (B) total carbonylated proteins and (C) Figure 6. Gene expression of markers of (A) proinflammatory
phospho-NFkB blots. Values are mean 6 SD; n = 4 replicates and (B) anti-inflammatory polarization from macrophages
from each group. *Significantly different from the PPP group treated with platelet-rich plasma (PRP) or platelet-poor
(P \ .05). plasma (PPP). Target gene expression was normalized to
b-actin. Values are mean 6 SD; n = 6 replicates from each
TNFa and NFkB pathways were the only 2 pathways that group. *Significantly different from the PPP group (P \ .05).
were predicted to be functionally activated in response to
PRP. Based on the role that the TNFa and NFkB pathways EGR2, and scleraxis are transcription factors known to
play in regulating ECM remodeling, oxidative stress, and play crucial roles in tendon development, growth, and
inflammation, we then chose to further evaluate and char- remodeling,16,20,36 and in the current study, PRP downre-
acterize these responses in greater detail. gulated the expression of all 3 of these genes. Tenomodu-
Both acute tendon tears and chronic degenerative tendi- lin, which is a marker of differentiated fibroblasts,20 was
nopathies involve a damaged or disordered ECM that must also downregulated by PRP. Autophagy is a catabolic cellu-
be remodeled and repaired by tendon fibroblasts.8,17 HA is lar process that is important in tissue remodeling,14,34 and
a glycosaminoglycan that serves as a template for new multiple genes that are important in the initiation and
ECM synthesis,3,36 and PRP treatment had no effect on maturation of autophagosomes were upregulated by PRP,
the expression of the major HA synthase enzymes HAS1 including Atg10, Bnip1, and GABARAPL2. The combined
and HAS2. PRP downregulated the expression of the major changes in ECM, MMP, tenogenesis, and autophagy gene
collagens in tendon, collagen 1 and 3, as well as elastin, expression suggest that PRP treatment likely actually
which has an important role in restoring ECM organiza- results in an atrophied ECM and reduced fibroblast activ-
tion after being stretched. In addition to these genes, sev- ity, which is largely consistent with a state of elevated
eral transcripts that help to assemble mature collagen acute tissue inflammation.8
fibrils, including the FACIT collagens 12 and 14, as well The TNFa/NFkB signaling cascade is a well-known
as CILP and fibromodulin, were downregulated after mediator of inflammation. Binding of TNFa to its receptor
PRP treatment. These results are in general agreement TNFR1 triggers activation of the IKK complex, which is
with collagen expression reported in previous work11,40 responsible for activating the p65 transcription factor sub-
and indicate that PRP treatment reduces the expression unit of NFkB through a combination of degradation of the
of major ECM components in tendon fibroblasts. PRP inhibitor IkB complex and phosphorylation of NFkB.2,35
also induced the expression of several major MMPs, includ- Once activated, NFkB translocates to the nucleus and
ing MMP3, MMP9, MMP10, and MMP13, which together induces the expression of numerous genes, many of which
degrade the major fibrillar collagens, minor collagens, are associated with inflammation. Oxidative stress is also
and other ECM structural proteins.8 While we are still in able to activate NFkB, often having an additive effect to
the early stages of understanding the networks of tran- TNFa signaling.27 In the current study, the treatment of
scription factors and signaling pathways that regulate ten- tendon fibroblasts with PRP resulted in an elevation of
don fibroblast specification and proliferation, EGR1, genetic markers of oxidative stress, including SOD1,
AJSM Vol. 44, No. 8, 2016 PRP Induces Oxidative Stress in Tendon Fibroblasts 1939

SOD2, NFE2L2, and Prdx1, as well as the chronic phos- M2c markers CD14, IL10, and CD163. With the exception
phorylation and activation of NFkB. Consistent with this, of VEGF, no macrophage phenotype marker that was eval-
we observed a marked increase in carbonylated proteins, uated showed tremendous changes in expression. In the
which are sensitive markers of elevated oxidative stress.30 absence of robust changes in more specific markers, we do
While TNFa is elevated in PRP and is able to induce oxida- not believe that VEGF alone can sufficiently mark the M1
tive stress through the induction of proinflammatory path- phenotype and conclude that PRP did not have a marked
ways,21 because platelets can produce and release effect on macrophage polarization. Interestingly, despite
hydrogen peroxide,13 it is possible that PRP also contains a substantial change in VEGF observed in macrophages,
endogenous peroxides that can produce ROS and further PRP treatment did not change VEGF expression in tendon
enhance oxidative stress in tendon fibroblasts. No change fibroblasts. Neovascularization is often observed in acute
in iNOS expression was observed, and combined with ele- and chronic tendon disorders,37,39 and it is possible that
vations in SOD1, SOD2, NFE2L2, and Prdx1, this suggests macrophages are the cells responsible for signaling of blood
that the elevated oxidative stress was likely caused by per- vessel ingrowth into damaged areas of tendons.
oxide-mediated processes instead of nitric oxide. Among This work has several limitations. These studies were con-
the more potent proinflammatory genes that are induced ducted in cultured rat cells, and while we attempted to sim-
in response to NFkB activation are the prostaglandin syn- ulate a native environment as much as possible in vitro, it is
thesis enzymes PTGES, Cox1, and Cox2.27 PRP treatment possible that cells would respond to PRP differently in vivo.
potently induced the expression of these 3 enzymes but had We also used a single dose of PRP added to culture media
no effect on 5-LOX expression, suggesting a role for prosta- and did not determine if there were dose-dependent effects
glandins but not leukotrienes in PRP-mediated inflamma- of PRP on cell behavior. For most measures, we evaluated
tion. PRP did not change the expression of SIRT1, which is changes in gene expression and not protein, and it is possible
an important and potent inhibitor of NFkB activity.21 Sev- that changes in gene expression do not result in changes in
eral other proinflammatory transcription factors were also protein levels. There are no commercially available PRP
upregulated by PRP treatment, including Fosb, Fosl1, and kits that have been validated for rats. Further, growth factor
c-Jun. These results together suggest that PRP treatment and cytokine levels in PRP can vary widely depending on the
induces a robust and heady induction of inflammatory and specific kit that is used.4 Despite these limitations, this study
oxidative stress pathways in tendon fibroblasts. provided important information related to the mechanism of
Macrophages appear to play an important role in the action of PRP in tendon fibroblasts and macrophages. Future
repair and regeneration of both acute tendon injuries and studies that use human cells or in vivo animal studies, pre-
chronic degenerative tendinopathies.7,9,39 Dragoo and col- pare PRP from commercial kits, use multiple doses and
leagues12 also observed that a PRP injection into otherwise time points, and analyze more changes at the protein level
healthy tendons resulted in an acute inflammatory response will provide further insight into the biology of PRP and hope-
and infiltration of macrophages into the injected tissue. This fully further refine its clinical use.
is consistent with findings in the current study, as PRP con-
tains elevated levels of several chemokine ligand proteins
that are involved in the recruitment of macrophages to tis- CONCLUSION
sue. CCL2 and CCL7 expression were also highly induced in
fibroblasts treated with PRP. Many of the individual compo- PRP has been used as a therapy for the treatment of ten-
nents of PRP are also able to polarize cultured macrophages don injuries and chronic diseases, but meta-analyses of
into a specific phenotype in isolation. For example, IFNg numerous clinical trials do not indicate a clear benefit for
and TNFa can prime macrophages to an M1 phenotype, the use of PRP in treating tendon disorders.25 A major rea-
while IL4 and IL10 are able to polarize macrophages to an son for this is the scarcity of trials that have enrolled large
M2 phenotype.28 There are no widely accepted and specific cohorts of patients as well as the substantial variation in
and definitive binary markers of the M1 or M2 phenotype, PRP preparation and delivery, patient demographics, and
and a panel of various markers and the fold change in these chronicity and site of injuries.25 Another limitation to the
markers are typically used to assess phenotypic changes in widespread acceptance of PRP for use in clinical practice
macrophage polarization.28,32,38 Within the M2 phenotype, is an inadequate understanding of its biological mecha-
there are subphenotypes, including M2a macrophages, nism of action. This study provided cellular and biochemi-
which are generally regarded as anti-inflammatory macro- cal data on the mechanism of action for PRP and reported
phages that function to resolve the M1 response, and M2c that it appears to work by inducing a massive inflamma-
macrophages, which function to promote tissue repair and tory reaction in tendon fibroblast cells. Inflammation is
regeneration.28 The specific markers for these subpheno- generally thought of in a negative fashion, but it also plays
types are less well defined than those that define M1 versus an important role in triggering a regeneration and repair
M2 macrophages; however, Arg1 and FGF2 are generally response.15 While the exacerbation of inflammation in an
considered M2a markers, while CD14, CD163, CD168, acute tendon injury may not be beneficial, inducing an
CD206, IGF1, IL10, and TGFb can mark M2c macro- acute bout of inflammation in chronic tendinopathies
phages.28,42 We observed that PRP treatment modestly may end up initiating a subsequent regenerative response.
increased the expression of the M1 markers iNOS and PRP is not unique in this manner; prolotherapy and needle
IL1b along with a robust increase in VEGF. However, there fenestration are used in the treatment of chronic tendino-
were also modest increases in the M2a marker Arg1 and pathies and have been proposed to work by a similar
1940 Hudgens et al The American Journal of Sports Medicine

mechanism of action.6 Considering the time and expense 19. Hsu WK, Mishra A, Rodeo SR, et al. Platelet-rich plasma in orthopae-
required in preparing PRP, future large clinical trials dic applications: evidence-based recommendations for treatment.
J Am Acad Orthop Surg. 2013;21(12):739-748.
that evaluate the ability of PRP to treat chronic tendon dis-
20. Huang AH, Lu HH, Schweitzer R. Molecular regulation of tendon cell
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mation and metabolic disorders. Cell Signal. 2013;25(10):1939-1948.
22. Kelly A, Lynch A, Vereker E, et al. The anti-inflammatory cytokine,
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