Current Concepts With Video Illustrations
Platelet-Rich Plasma: A Milieu of Bioactive Factors
Stacie G. Boswell, D.V.M., Brian J. Cole, M.D., M.B.A., Emily A. Sundman, B.S.,
Vasili Karas, B.S., and Lisa A. Fortier, D.V.M., Ph.D.
Abstract: Platelet concentrates such as platelet-rich plasma (PRP) have gained popularity in sports
medicine and orthopaedics to promote accelerated physiologic healing and return to function. Each
PRP product varies depending on patient factors and the system used to generate it. Blood from some
patients may fail to make PRP, and most clinicians use PRP without performing cell counts on either
the blood or the preparation to confirm that the solution is truly PRP. Components in this milieu have
bioactive functions that affect musculoskeletal tissue regeneration and healing. Platelets are activated
by collagen or other molecules and release growth factors from alpha granules. Additional substances
are released from dense bodies and lysosomes. Soluble proteins also present in PRP function in
hemostasis, whereas others serve as biomarkers of musculoskeletal injury. Electrolytes and soluble
plasma hormones are required for cellular signaling and regulation. Leukocytes and erythrocytes are
present in PRP and function in inflammation, immunity, and additional cellular signaling pathways.
This article supports the emerging paradigm that more than just platelets are playing a role in clinical
responses to PRP. Depending on the specific constituents of a PRP preparation, the clinical use can
theoretically be matched to the pathology being treated in an effort to improve clinical efficacy.
have gained popularity in fields such as wound heal-
ing,1dental and maxillofacial surgery,2sports medi-
cine,3and veterinary medicine.4In sports medicine
there are numerous clinical objectives motivating the
use of PRP, including promotion of tissue regenera-
tion in both bony5,6and soft tissues,7prevention or
reparations of platelet concentrates are generi-
cally referred to as platelet-rich plasma (PRP) and
treatment of infection,8,9and restoration of function.10
Clinical observation and opinion suggest that pain
relief and return to function occur more rapidly than
expected for some healing orthopaedic problems after
the use of PRP. This has led to investigations of
antinociceptive properties of PRP in our laboratory
and others.11In addition to being evaluated in vivo for
efficacy and safety, in vitro investigation of PRP and
the growth factors (GFs) contained within it has been
Generation of PRP is accomplished with one of
many available commercial systems that are marketed
primarily based on their ability to concentrate plate-
lets. Targeted musculoskeletal tissues, such as tendon,
ligament, and cartilage, heal slowly because of a lim-
ited blood supply, slow cell turnover, and limited
extracellular matrix restoration.12PRP provides GFs
that stimulate neovascularization and increase the
blood supply and nutrients needed for cells to regen-
erate the damaged tissue (Fig 1). Neovascularization
also brings new cells and removes debris from dam-
aged tissue. It is hypothesized that the GFs released
From the Department of Clinical Sciences, College of Veterinary
Medicine, Cornell University (S.G.B., E.A.S., L.A.F.), Ithaca, New
York; and Midwest Orthopedics at Rush, Rush University Medical
Center (B.J.C., V.K.), Chicago, Illinois, U.S.A.
The authors report that they have no conflicts of interest in the
authorship and publication of this article.
Received June 13, 2011; accepted October 19, 2011.
Address correspondence to Lisa A. Fortier, D.V.M., Ph.D., Cornell
University, Ithaca, NY 14853, U.S.A. E-mail: email@example.com
© 2012 by the Arthroscopy Association of North America
Note: To access the videos accompanying this report, visit the
[Month] issue of Arthroscopy at www.arthroscopyjournal.org.
Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol xx, No x (Month), 2012: pp xxx
leukocytes such as neutrophils and monocytes that mainly function in phagocytosis, immunity, and inflammation. Soluble hormones (such
as IGF-1) are also shown. Cells in the joint that could be exposed to the effects of the bioactive factors in PRP include synovial, meniscal,
and ligamentous fibroblasts, chondrocytes, and osteocytes. Green solid arrows indicate positive metabolic paths (such as upregulation of
matrix synthesis or cell replication). Red dashed arrows indicate negative metabolic paths (such as increased matrix degradation or inhibition
of matrix synthesis). (ADP, adenosine diphosphate; CaCl2, calcium chloride; ECM, extracellular matrix; EGF, epidermal growth factor; IL-1,
interleukin 1; IL-6, interleukin 6; ligs, ligaments; TNF-?, tumor necrosis factor ?; VEGF, vascular endothelial growth factor; vWF, von
Schema of PRP injection into patellofemoral joint. Cells in PRP include platelets, which are activated to release GFs, and
S. G. BOSWELL ET AL.
from platelets in PRP accelerates integration of bio-
logic grafts and healing so that patients can return
more rapidly and functionally to activities.12,13
Recently, there has been an increased focus on other
components of PRP, particularly leukocytes14and fi-
brinogen.15Classification or characterization of PRP
based on platelet and white cell counts alone has been
attempted.15This brings some uniformity to the PRP
field and improves the specificity of comparative in-
vestigations of PRP. However, PRP is best considered
a milieu of bioactive factors, and the resultant blend of
these factors will determine the most relevant appli-
cations to maximize clinical outcomes.
The purpose of this article is to consider the bioac-
tive cellular and molecular factors in PRP and sum-
marize what is known regarding the effect of each
factor on musculoskeletal tissue healing. The clinical
application of PRP has been reviewed elsewhere and
therefore is not a focus of this report.13,16
PRP is a plasma suspension that contains all com-
ponents of whole blood in varying amounts (Videos 1
and 2, available at www.arthroscopyjournal.org). Ac-
cording to the Red Cross, PRP by definition contains
a minimum of 200,000 platelets/?L. Preparation pro-
cesses take advantage of differing density gradients of
the components in blood to concentrate platelets.
Centrifugation of whole venous blood containing an
anticoagulant results in a plasma supernatant with a
gradient of cellular concentration. Erythrocytes are
the densest and will remain as the packed cell layer
at the bottom of the centrifuge container. The buffy
coat of white blood cells is at the top of the packed red
blood cell layer. The platelets are at the highest concen-
tration in the plasma just above the buffy coat and
decrease in concentration toward the top of the plasma
reduces the number of erythrocytes and second con-
centrates platelets. The various systems differ in
platelet collection efficiency and repeatability, final
leukocyte count, platelet activation, and ease of
Differences between PRP preparations can be due
to the proprietary system selected or the many other
factors that also affect the final product. Peripheral
venous blood parameters influence the contents of the
final PRP product. For example, platelet count in the
final concentrate is dependent on the whole blood
platelet count in a linear manner.18Hematocrit also
influences the final product, especially in fixed-volume
separator techniques. These types of systems have a
requisite volume of whole blood, which is centrifuged
before removal of a predetermined volume of the
packed cells and/or plasma layer. Therefore a variable
number of erythrocytes remain in the PRP. Storage of
whole blood before processing introduces additional
variability of the final PRP and platelet characteristics
There is variability in the number of platelets and
leukocytes in PRP between preparations from the
same individual that is not system dependent. Both the
absolute and relative numbers of each leukocyte type
change compared with those found in peripheral blood
and are variable with the proprietary system selected.
This variation can be partially attributed to a number
of factors, including hydration status, inflammation
(leukocytosis or leukopenia), lipemia (which increases
platelet concentration and is influenced by diet),21or
circadian rhythms in platelet numbers.22-24Although
gender does affect hematocrit, it affects the final PRP
less if a density-gradient system is used. An investi-
gation using cultured periodontal osteoblasts found
that the gender of the blood donor for generation of
PRP did not significantly alter either platelet count or
We have observed that some individuals can have a
complete failure to concentrate platelets with one sys-
tem but are successful in concentrating platelets with
a system from a different manufacturer (Table 2). This
disparate result can occur with one blood draw allo-
cated for use in 2 systems, indicating that failure to
generate PRP is possible in any individual and when
using any system. These observations suggest that a
complete blood count should be performed on each
patient’s venous blood and PRP so that a clinician can
be sure that the patient is truly being treated with PRP.
Such complete blood count data would also facilitate
determination of which PRP components most affect
Key Points of PRP Processing
PRP contains all components of blood in variable quantities.
A final concentration of ?200,000 platelets/?L meets the Red
Cross definition of PRP.
The final PRP product may differ because of the proprietary
system selected for processing.
PRP is affected by the patient’s venous blood status (packed cell
volume, hydration, medications, circadian rhythms).
A clinician’s awareness of exactly what is contained in PRP will
allow a better-informed decision to be made regarding its
BIOACTIVE FACTORS IN PRP
Platelets (thrombocytes) range from 2 to 3 ?m in
size while circulating for 7 to 10 days at concentra-
tions of 150 to 400 ? 103/?L. They are anucleate
cytoplasmic fragments of multinucleated megakaryo-
cytes located in bone marrow. Platelets are most often
thought of primarily for their hemostatic and coagu-
lation functions; however, proteomic studies have
shown that platelets contain over 800 proteins with
numerous post-translational modifications, such as
phosphorylation, resulting in over 1,500 protein-based
bioactive factors.26,27Only some of these proteins’
physiologic actions have been studied, including GFs,
peptide hormones, and chemoattractants for macro-
phages, neutrophils, and stem cells.
Circulating, inactive platelets have a discoid
shape with an open canalicular system. Both native
and exogenous molecules can activate platelets, in-
cluding collagen, platelet-activating factor, serotonin,
calcium, magnesium, thromboxane A2(TXA2), aden-
osine diphosphate (ADP), and thrombin. In a positive-
feedback system, activated platelets release TXA2,
ADP, and thrombin and activate other nearby plate-
lets. Facilitated by actin and myosin filaments, the
activated platelet undergoes cytoskeleton restructuring
to develop multiple filopodia from the location of the
canaliculi. Exocytosis and degranulation result in an
overall increase of platelet surface area. In vitro ob-
servations have shown that when platelets are acti-
vated, an initial burst of GF release is followed by
further sustained release.28Platelet activation results
in an increase in anti-inflammatory cytokines because
of the presence of hepatocyte GF.29
In addition to platelet activation by endogenous
chemokines, activation is accelerated by adrenergic
activity,30oxidative stress,31or chemical use, such as
smoking.30Platelets are heterogeneous in size. Larger
platelets from healthy volunteers have been shown to
be more active and release more chemokines than
smaller platelets.32Perhaps most significantly, in vitro
experiments have shown that platelets in PRP are
activated by bone substitution materials33and biphasic
osteochondral scaffolds.28Platelet aggregation is de-
creased by a strenuous workload34or substances such
as caffeine35or propofol.36
When the concentration of GFs is measured, PRP
preparations typically contain a 3- to 5-fold increase
compared with baseline.37This increase is attributed
to both platelet concentration and activation. Sev-
eral studies have investigated the effects of platelet
concentration on musculoskeletal tissue homeosta-
sis.4,13,14,38Schnabel et al.4showed that a concen-
trated platelet preparation resulted in an enhanced
anabolic gene expression in tendon and ligament. In
clinical equine tendonitis, a concentration of 750,000
platelets/?L in PRP was significantly associated with
White Blood Cell and Platelet Counts in PRP From Healthy Volunteers,
Showing Failed PRP Production
(cells ? 103/?L)
(cells ? 103/?L)
(cells ? 103/?L)
(cells ? 103/?L)
Individual 2 4.3
Individual 3 5.6
Individual 4 7.5
Abbreviations: Mfr, manufacturer; NA, not available; Plt, platelet count; WBC, total white blood
*These 6 individuals had a reduced number of platelets compared with venous blood in at least one of
their PRP products, despite an increase in platelet count with another manufacturer’s production system.
This suggests that clinicians should confirm platelet counts in PRP to ensure that the expected concen-
tration has occurred during processing.
†PRP production on a different day from other values for the same individual.
S. G. BOSWELL ET AL.
a shorter time to recovery, defined as return to race
competition.38However, a ceiling threshold of benefit
from GFs seems to exist. One experiment described an
increase in chondrocyte proliferation with addition
of recombinant platelet-derived growth factor-BB
(PDGF-BB) in culture media. The concentration used
varied from 4.7 to 300 ng/mL, but peak proliferation
was noted at 75 ng/mL.39In a different part of the
same study, PDGF-AB was used to stimulate migra-
tion of bovine meniscal cells with a maximum effect
at 10 ng/mL and inhibition of chemotaxis occurring at
higher concentrations.39An upper threshold of benefit
could occur in vivo if a large volume is injected into
a small lesion.
In addition to releasing GFs, some literature sup-
ports the concept that PRP regulates local production
of these factors. Using PRP in a rabbit model of
Achilles tendon healing, Lyras et al.40showed an
upregulation of intracellular transforming growth fac-
tor (TGF) ?1in the first 2 weeks of healing with
downregulation in the third and fourth week of healing
compared with controls. Furthermore, an increase of
intracellular insulin-like growth factor 1 (IGF-1) ex-
pression was shown in tenocytes throughout the 4
weeks of healing.41
Platelet alpha granules are 300- to 500-nm mi-
crovesicles with a proteome count of approximately
284.42These include bioactive molecules such as ad-
hesive proteins (fibrinogen, von Willebrand factor)
and receptors, clotting factors (V, XI, XIII, and pro-
thrombin), fibrinolytic factors (antithrombin, plasmin,
and plasminogen), other basic proteins, membrane
glycoproteins, and GFs (Table 3).8Alpha granules
give platelets their characteristic purple granular ap-
pearance on a typical blood smear. GF peptides re-
leased from alpha granules include PDGF, TGF-?1,
vascular endothelial growth factor, basic fibroblast
growth factor (bFGF), and epidermal growth factor.
Despite reports in earlier literature, IGF-1 is not stored
in platelets but it is in plasma. Individual variation in
the concentration of GFs released from granules ex-
ists, but the platelet number in PRP is positively
correlated with GF concentration.12,14,43PDGF is in-
tegral to cell proliferation, chemotaxis, cell differen-
tiation, and angiogenesis.39,44For example, Marko-
poulou et al.25determined that PRP promoted cellular
proliferation of human osteoblasts that was due to
PDGF, bFGF, and TGF-?1. Increased cellular prolif-
eration is one way that PRP promotes healing of
musculoskeletal tissues. Chung et al.44showed that
blocking PDGF-BB in a rodent growth plate damage
model resulted in reduced chemotaxis of mesenchy-
Modulation of coagulation and vascular repair by
GFs in PRP is theorized to result in accelerated and
improved wound, tendon, ligament, and bony heal-
ing.12Extensive reviews on the use and effects of GFs
from alpha granules on musculoskeletal tissues are
GFs function by binding to cellular transmembrane
receptors and regulating cellular signaling pathways.47
One advantage of PRP over administration of a single
exogenous GF is that GFs are released from platelets in
native (rather than recombinant) form and presumably in
a biologically relevant ratio.37In a canine model, use of
bFGF alone accelerated the cell-proliferation phase of
tendon healing but resulted in peritendinous scar for-
mation and diminished range of motion.48This finding
Components of PRP Relevant to Musculoskeletal Tissue
Summary of Cellular and Molecular
ProteinsAlbumin, globulins, fibrinogen,
complement, and clotting factors
Chloride, sodium, potassium, and
IGF-1, estrogens, progesterone,
androgens, ACTH, and HGH
COMP, CD11b, protein C,
microRNA, osteocalcin, and
Alpha granulesAdhesive proteins, clotting factors,
and GFs PDGF, TGF-?, VEGF,
FGF, EGF, and HGF
Calcium and neurotransmitters
Primary granulesMyeloperoxidase, acid hydrolases,
defensins, and serine proteases
phagocytins, and lysozyme
Gelatinase and proteases
Platelet-activating factor, TGF-?,
VEGF, FGF, and EGF
ATP, S-nitrosothiols, nitric oxide,
hydrogen sulfide, hemoglobin,
and free radicals
Abbreviations: ACTH, adrenocorticotropic hormone; ATP,
adenosine triphosphate; COMP, cartilage oligomeric matrix pro-
tein; EGF, epidermal growth factor; FGF, fibroblastic growth fac-
tor; HGF, hepatocyte growth factor; HGH, human growth hor-
mone; VEGF, vascular endothelial growth factor.
BIOACTIVE FACTORS IN PRP
supports the concept of using a balanced, complemen-
tary set of bioactive GFs found in PRP, rather than
individual GFs. PRP is also much less expensive and
subject to fewer regulatory restrictions than use of a
Dense bodies (delta granules) are organelles 250 to
300 nm in size. They contain primarily substances that
promote clotting, such as calcium (clotting factor IV),
magnesium, adenosine, serotonin, and histamine.47A
deficiency of delta granules results in mild bleeding
disorders. Although serotonin is manufactured in the
gastrointestinal tract and the brain, it is stored in dense
bodies and, when released, promotes hemostasis by
constricting vascular tone and permeability. Details
are still not fully elucidated, but it is known that
serotonin exists in different forms and that it acts as
either a hormone or neurotransmitter and can both
positively and negatively regulate bone mass.50
The lambda granule is the third type of granule
whose contents are released during platelet activation.
Lambda granules are lysosomal-type organelles. Al-
do have “clearing” responsibilities to remove infectious
agents and cellular debris. As healing progresses, tissue
plasminogen activator secreted by the endothelium acti-
vates lambda granule enzymes, which convert plasmin-
ogen to plasmin and lyse the clot. Plasminogen activa-
tors play a role in homeostasis of muscle fibers and
adjoining extracellular matrix, including fracture
Plasma is defined as the yellow-colored liquid com-
ponent of blood in which blood cells are suspended.
Approximately 200 proteins have been documented in
plasma, including albumin, immunoglobulins, com-
plement, and clotting factors.53Because plasma tran-
siently contains many proteins released from cells and
metabolic processes throughout the body, the presence
of as many as 679 proteins has been documented.54
Biomarkers of bone turnover can be found in plasma
as well, including osteocalcin and osteonectin, which
are both secreted by osteoblasts. Other musculoskel-
etal molecules of interest are transiently found in
plasma. For example, cartilage oligomeric matrix pro-
tein levels are elevated in cases of severe arthritis.55
Plasma biomarkers can be measured during orthopae-
dic surgery. Hughes et al.56measured markers of
ongoing orthopaedic-specific inflammation and leuko-
cyte activation including nonspecific C-reactive pro-
tein and orthopaedically related CD11b and protein C
and showed that more accurate assessment of intraop-
erative inflammation was feasible. MicroRNAs pres-
ent in plasma have been used to document both rheu-
matoid arthritis and osteoarthritis.57A biomarker
profile has been used to document high bone turnover
in women with osteoporosis.58A similar profile could
be developed for patients with fractures. Likewise, an
equine model showed that a biomarker pattern was
present in racehorse plasma before injury.59
Plasma proteins involved in hemostasis also affect
musculoskeletal healing. After the activated platelet
plug provides primary hemostasis at a site of injury
and during clot maturation, cell adhesion molecules
such as fibronectin, fibrin, and vitronectin move from
the plasma into the clot. In vitro, these proteins have
been shown to induce chemotaxis of multipotent stro-
mal cells across a membrane.60This implies that these
molecules may modulate cell migration of fibroblasts,
osteoblasts, or other tissue-regenerating cells in mus-
Electrolytes present in plasma are tightly regulated
through transmembrane adenosine triphosphatases to
optimize cellular and tissue function. Chloride (refer-
ence interval, 340 to 370 mg/dL), sodium (31 to 35
mg/dL), potassium (14 to 20 mg/dL), and total cal-
cium (8.5 to 10.2 mg/dL) are the 4 electrolytes that are
About half of the calcium circulating is bound by
albumin, and ionized calcium can be measured inde-
pendently (4.1 to 4.8 mg/dL). In addition to function-
ing as a platelet activator, intracellular reservoirs
maintain calcium homeostasis. It is an important sec-
ondary messenger in cells and is a cofactor for several
extracellular reactions. It has been well established
that within the coagulation cascade, plasma calcium is
a cofactor for the formation of both the tenase (factor
X) and prothrombinase complexes. In relation to mus-
culoskeletal tissue repair, intracellular calcium signal-
ing is required for the contractile activity of myofi-
broblasts in vitro.61
Hormones such as thyroxine, estradiol, adrenocor-
ticotropic hormone, androgens, estrogens, progester-
one, and human growth hormone circulate in plasma.
Perhaps the most musculoskeletally relevant hormone
found in plasma is IGF-1. IGF-1 has been shown to
improve healing in equine tendon and cartilage mod-
els.62,63The benefits of IGF-1 in enhancing regenera-
tion of injured musculoskeletal tissue has been previ-
How much the other hormones present in PRP
affect platelets or musculoskeletal metabolism is not
fully known. In general, glucocorticoids provide pro-
S. G. BOSWELL ET AL.
tection to tissues by decreasing the production or
activity of inflammatory mediators. Intracellular syn-
thesis of epidermal growth factor is stimulated by
testosterone and inhibited by estrogens. Experimen-
tally induced platelet aggregation and release of TXA2
were reduced in PRP with a physiologic level of
dihydrotestosterone.31Estrogens alone do not appear
to affect platelet aggregation in PRP; however, some
estrogens with ADP or adrenaline synergistically in-
crease platelet aggregation.65
Mammalian leukocytes are classified as granulo-
cytes (neutrophils, eosinophils, and basophils) or
mononuclear cells (lymphocytes and monocytes or
macrophages). Phagocytic cells such macrophages are
essential to the in vivo healing process.66A murine
model was used to show that macrophages were es-
sential for debridement of damaged ligamentous tissue
and for cytokine release that mediates the repair pro-
cess.66The presence of leukocytes can result in pro-
inflammatory cellular signaling and local tissue catab-
olism.67For example, McCarrel and Fortier14showed
that leukocytes in a PRP preparation had a negative
correlation with matrix synthesis and a positive cor-
relation with matrix catabolism in tendons. A higher
concentration of leukocytes, therefore, is likely unde-
sirable for musculoskeletal applications associated
with tendon healing but could be more relevant for
other uses, such as healing of large, infected dermal
The primary function of a neutrophil is to destroy
infectious agents. Reservoirs of antimicrobial proteins
are contained in primary, secondary, and tertiary gran-
ules. Proteases are also found in the cytoplasm of
neutrophils. Although these molecules are important
for destroying microbes, they can also incite local
If the goal of PRP is to provide balanced yet aug-
mented healing, adding neutrophils in excess is antag-
onistic to the goal. To prevent neutrophil-mediated
tissue damage, the influx of neutrophils must be con-
trolled.68The quantity of neutrophils in the native
environment is self-controlled. Secretory leukocyte
protease inhibitor is synthesized in myelocytes, stored
in neutrophils, and released to limit local tissue dam-
age caused by serine proteases.67In addition, neutro-
phils release reactive oxygen species, which results in
a respiratory burst that is intended to damage patho-
gens. However, the respiratory-burst reaction without
a target microbe is likely to cause tissue damage.13
Primary (azurophilic) granules are smaller and more
numerous than secondary granules and contain at least
10 peptides with antimicrobial properties. When a
neutrophil is activated, the primary granules release
myeloperoxidase, lysozyme, acid hydrolase, alpha de-
fensins,68bactericidal/permeability increasing protein,
and serine proteases (including neutrophil elastase
[ELA2], azurocidin, cathepsin G, and proteinase 3).67
These proteases both damage bacteria and degrade the
extracellular matrix, allowing cellular migration through
the tissue.69,70Although these functions are essential for
tissue remodeling, they can also result in destruction of
normal tissue, as exemplified in an in vitro study using
normal equine tendons.14
Secondary (specific) granules are less numerous and
larger than primary granules. Some molecules carried
by primary granules are also carried by secondary
granules (lactoferrin, gelatinase, lysozyme).71In addi-
tion, secondary granules contain matrix metalloprotei-
nase-8 (MMP-8) (neutrophil collagenase) and MMP-9
(type IV collagenase, also known as gelatinase B),
which result in extracellular matrix degradation.13
They also contain the antimicrobial polypeptides ly-
sozymes, lactoferrin, and cathelicidin. Secretory leu-
kocyte protease inhibitor is also present and modulates
the neutrophil’s proteolytic response in any tissue.67
Lactoferrin binds iron and is active against bacteria,
viruses, fungi, and parasites.69Its antiviral action oc-
curs through competitive binding of glycosaminogly-
cans, preventing viral adsorption and cellular invasion.
Thus it could also block glycosaminoglycans found in
cartilage, tendon chondroitins, or synovial hyaluronan.
Tertiary granules are both smaller and less numer-
ous than other granule types. They release MMP-9
and MMP-15 along with peptides that sequester iron
and other metals, including neutrophil gelatinase–
associated lipocalin and natural resistance–associated
macrophage protein 1, from phagocytosed bacteria.70
Monocytes are found in peripheral blood and dif-
ferentiate into macrophages when they migrate into
connective tissue. Circulating monocytes promote ex-
tracellular matrix breakdown by the release of
MMP-2, MMP-9, MMP-13, cathepsin, inducible nitric
oxide synthase, interleukin-1, and interleukin-6. Con-
currently, monocytes suppress inflammation, promote
angiogenesis, and support collagen synthesis through
TGF-?, vascular endothelial growth factor, and bFGF
release, respectively. Monocyte-derived macrophages
are necessary for any type of regenerative process,
including musculoskeletal tissue repair through active
phagocytosis of necrotic tissue and debris. Activated
BIOACTIVE FACTORS IN PRP
macrophages likely play a role in the regeneration of
Eosinophils function mainly in immunity. They
contain major basic protein 1, which is also found in
basophils and mast cells and is involved with killing
endoparasites. Eosinophil peroxidase is only found in
eosinophils and inactivates pathogens through oxida-
tion. Ribonucleases from eosinophils are antiviral and
The centrifugation process used to generate PRP
typically reduces or eliminates the presence of eryth-
rocytes (red blood cells). Because some red blood
cells are often present, they are worth discussing. The
main function of erythrocytes is to carry oxygen,
which is essential for tissue repair. As such, red blood
cells lack most cellular organelles, including a nu-
cleus, endoplasmic reticulum, and mitochondria. They
contain about 750 proteins compared with the esti-
mated 20,000 or more proteins of nucleated cells.73
Red blood cells also carry some immune complexes.74
In vivo, erythrocytes release substances that serve to
dilate vessels including adenosine triphosphate, S-
nitrosothiols, nitric oxide, and hydrogen sulfide. Ex-
periments suggest that nitric oxide mediates insensi-
tivity to IGF-1 in diseased cartilage.75,76Hemoglobin
carries the oxygen and, technically, is a metallopro-
teinase. It is made of 4, smaller, protein-bound heme
molecules. Under conditions of oxidative stress, heme
can become free and cytotoxic. The iron contained
within heme molecules catalyzes free radicals and
contributes to pathogen destruction. The free radicals,
however, can also induce apoptosis of host cells in
reaction to proinflammatory signaling. Because of
these destructive capabilities, limiting red blood cell
contamination in a PRP preparation intended for treat-
ment of musculoskeletal repair seems warranted.
Knowing the components of PRP being used will
help elicit the important factors in various applications
of this regenerative therapy (Table 4). As an example,
5 recent articles in Arthroscopy evaluated anterior
cruciate ligament reconstruction and compared PRP-
augmented procedures with controls.77-81Magnetic
resonance imaging evaluation times ranged from 3 to
24 months postoperatively, and results of these trials
were variable. Several letters to the editor and the
authors’ replies highlight the controversy and confu-
sion regarding PRP use in these trials and emphasize
the need for more Level I trials to determine the
efficacy of PRP.82-85Of the reports, one included
platelet counts in its results and none described leu-
kocyte counts of the PRP. Four of 5 did state which
proprietary system was used to produce the PRP used
in clinical patients—all of which were different. Un-
derstanding what is in PRP is necessary to evaluate
why it is or is not efficacious, which patients will
receive the most benefit, and when it will provide the
PRP is a useful regenerative therapy to address
many musculoskeletal injuries. It is important to un-
derstand that PRP is more than just platelets and that
it contains many bioactive factors that act in anabolic,
catabolic, proinflammatory, and anti-inflammatory
pathways. Some components are also involved in the
modulation of the immune response. The precise com-
bination and concentration of platelets, leukocytes,
and other plasma components best for musculoskeletal
healing are not presently known, and clinicians should
be aware that the effects of PRP are not solely based
on platelet concentration. A maximal efficacious con-
centration beyond which the platelet concentration
will provide no further clinical benefits likely exists.
Although the effects of many of the proteins in PRP
on musculoskeletal tissues are still unknown, they
likely contribute to the biologic healing process. Fi-
nally, it is imperative for individuals involved in clin-
ical study design and all clinicians to take into con-
sideration diurnal variation in platelet count and that,
simply, generation of PRP will fail in some patients in
some instances. Although PRP is more than just plate-
lets, the clinician should confirm that PRP, by its very
definition, has been generated from peripheral blood
Tips for PRP Use
Perform a CBC on venous blood and PRP to confirm that the
product intended for use is truly PRP.
Always maintain the sterility of the PRP.
When injecting PRP, do not exceed the volume limit of the
Handle PRP gently to avoid premature platelet activation.
Consider individual, diurnal, and medication-influenced factors
when selecting a time to make PRP.
Abbreviation: CBC, complete blood count.
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