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Nicola Maffulli

Editor

Platelet Rich Plasma


in Musculoskeletal
Practice

123
Platelet Rich Plasma
in Musculoskeletal Practice
Nicola Maffulli
Editor

Platelet Rich Plasma


in Musculoskeletal
Practice
Editor
Nicola Maffulli
Department of Musculoskeletal Disorders
Faculty of Medicine, Surgery and Dentistry
University of Salerno
Salerno
Italy

ISBN 978-1-4471-7270-3 ISBN 978-1-4471-7271-0 (eBook)


DOI 10.1007/978-1-4471-7271-0

Library of Congress Control Number: 2016947466

© Springer-Verlag London 2016


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Preface

In the near future, most Western countries will have to cope


with the economic implications of the demands imposed by
the growing rate of musculoskeletal ailments. Therefore, there
is a real need to develop further research and novel therapies.
Osteoarticular conditions and tendinopathies affect both the
elderly and young adults, are common, and cause severe long-
term pain and chronic impairment. These impact negatively
on the quality of life and induce sedentarism.
The problem is worsening, as there is an increased inci-
dence of the risk factors, including ageing, traumatic sports
injuries, and metabolic diseases. Morbidity and financial
costs are of great consequence. Critical progress on novel
biological interventions could meet pressing health needs,
reduce costs, and enable patients to resume an active healthy
lifestyle.
Over the past decade, the concept that chronic musculo-
skeletal conditions such as osteoarthritis and tendinopathy
are linked to deficient healing by failure of one or several of
the cellular/molecular processes involved has gained ground.
The economic and social burden of musculoskeletal disor-
ders is challenging, and biological interventions in their pres-
ent form cannot meet the clinical demand for long-lasting
treatment.
Novel technologies are making it possible to deal with the
intrinsic complexity of regenerative mechanism. However,
technologies aimed at this are still expensive and limited.
In this context, the use of platelet rich plasma (PRP) and
its derivatives is encouraging. Blood and its products are
v
vi Preface

appealing: blood contains biologically active factors, and it


is responsible for haemostasis, synthesis of new connective
tissue, and revascularization. Growth factors, and the induc-
tion of further release of growth factors, seem to improve
the healing process in chronic injuries and accelerate repair
in acute and chronic lesions. PRP can be used to enhance
articular and soft tissue healing and is often recommended
as best practice for management of musculoskeletal injuries.
There are still many unanswered questions about the most
appropriate volume and frequency of the injections, the ideal
period between multiple injections, and the mechanism by
which the beneficial effect would be harnessed. In this con-
text, this book offers some insights in the use of PRP in this
field. It is not exhaustive and is written by enthusiasts who
have performed much research on the topic. Above all, all
the authors acknowledge that we are at the beginning of a
long road ahead of us. We still do not know why PRP works,
when it is contraindicated, and why some patients respond to
it wonderfully well, or not at all. We have to start, however,
and this is the first step.

Salerno, Italy Nicola Maffulli


London, UK
About the Editor

Nicola Maffulli Department of Musculoskeletal Disorders,


Faculty of Medicine, Surgery and Dentistry, University of
Salerno, Salerno, Italy
Centre for Sports and Exercise Medicine, Barts and London
School of Medicine and Dentistry, Queen Mary University of
London, London, UK

vii
Contents

1 Contents and Formulations of Platelet


Rich Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Amy S. Wasterlain, Hillary J. Braun,
and Jason L. Dragoo
2 Platelet-Rich Plasma in Pain Medicine . . . . . . . . . . . 31
José Fábio Santos Duarte Lana,
Eduardo Fonseca Vicente, Adam Weglein,
William Dias Belangero, Fabrício Dias Assis,
and André Marques Mansano
3 PRP: Tips for Application
in the Musculoskeletal System . . . . . . . . . . . . . . . . . . . 63
Steven Sampson, Ken Mautner, Alessio Giai Via,
and Angie Botto-van Bemden
4 PRP in Tendons and Other Non-bone Tissues . . . . . 93
Sebastiano Vasta, Rocco Papalia,
Vincenzo Denaro, and Nicola Maffulli
5 Platelet Rich Plasma in Articular
Cartilage Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Elizaveta Kon, Giuseppe Filardo,
Berardo Di Matteo, Giulia Venieri,
and Maurilio Marcacci
6 Platelet-Rich Plasma in Knee Osteoarthritis
in the Athlete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Mary Alexis Iaccarino and Joanne Borg-Stein
7 Platelet Rich Plasma in Foot and Ankle Surgery . . . 147
Catie Cunningham, Amit Sood, and Sheldon Lin
ix
x Contents

8 Platelet Rich Plasma for Biological Therapy:


Applications and Limits . . . . . . . . . . . . . . . . . . . . . . . . 175
Giuliana Gobbi and Marco Vitale
9 The Systemic Effects of Platelet-Rich Plasma. . . . . . 199
Amy S. Wasterlain, Hillary J. Braun,
and Jason L. Dragoo
10 Potential Links Between Tendon Pathology
and Platelet Rich Plasma Biology . . . . . . . . . . . . . . . . 223
Isabel Andia, Eva Rubio-Azpeitia,
and Nicola Maffulli

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Chapter 1
Contents and Formulations
of Platelet Rich Plasma
Amy S. Wasterlain, Hillary J. Braun, and Jason L. Dragoo

Multiple studies have demonstrated a role for platelet-rich


plasma (PRP) in accelerating and facilitating response to
injury. The cellular response to injury progresses through four
general stages: hemostasis, inflammation, proliferation, and
finally remodeling. Each phase is characterized by enhanced
cellular or molecular activity, all of which involve platelets.
Blood plasma and platelets are responsible for hemostasis,
while leukocytes and activated platelets mediate inflamma-
tion, and growth factors derived from platelet α-granules
influence tissue regeneration. Specifically, the leukocyte con-
tent of PRP is thought influence the inflammatory phase,
while angiogenic and mitogenic growth factor concentrations
are believed to aid tissue regeneration [1]. Both the precise
composition and formulation of PRP affect the cellular envi-
ronment in which it is placed and determine its overall effect
on tissue repair.

A.S. Wasterlain, MD • H.J. Braun, MD • J.L. Dragoo, MD ()


Department of Orthopaedic Surgery, Stanford University,
Stanford, CA, USA
e-mail: jdragoo@stanford.edu

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 1


Practice, DOI 10.1007/978-1-4471-7271-0_1,
© Springer-Verlag London 2016
2 A.S. Wasterlain et al.

Content of Platelet-Rich Plasma Preparations


The goal of any tissue regeneration therapy is to facilitate the
development of a well-organized extracellular matrix capable
of attaining the mechanical performance and functionality of
non-injured tissue [2]. Therefore, it is imperative to character-
ize the molecular constituents of these therapies when evalu-
ating their efficacies. The cellular response to PRP is
influenced by the composition of the PRP, including the rela-
tive concentrations of platelets, white blood cells (WBCs),
fibrinogen and fibrin, and growth factors.

Platelets

Currently, PRP is consistently defined only by the absolute


quantity of platelets, and not by other components. In
humans, the normal platelet count in whole blood ranges
from approximately 150,000 to 350,000/μL [3], whereas plate-
let-rich plasma is often defined as at least 1,000,000 platelet/
μL suspended in plasma [4]. In order to ensure that the plate-
lets are suspended and do not form a clot, PRP must be made
from anticoagulated blood.
The logic behind PRP is that platelets are the first to arrive
at the site of tissue injury, and thus have the potential to
release growth factors that play a critical role in mediating
healing [5]. For example, skin, synovium and tendon treated
with plasma preparations containing two- or fourfold platelet
concentrations showed significantly more cellular prolifera-
tion than untreated tissues [6]. Many of the cytokines and
growth factors believed to be responsible for the effects of
PRP are contained within the α-granules of platelets. Basic
cytokines contained within platelets include insulin-like
growth factor (IGF-1), transforming growth factor-β (TGF-
β), platelet-derived growth factor (PDGF), fibroblast growth
factor (FGF), epidermal growth factor (EGF), and vascular
endothelial growth factor (VEGF) [3]. Platelet activation
triggers degranulation and release of these growth factors.
Chapter 1. Contents and Formulations 3

The timing and cumulative release of growth factors is deter-


mined by the activation method (see “Platelet Activation”
below), but may continue throughout the platelet’s 8–10 day
lifespan [3]. Many experts have suggested that in contrast to
exogenous growth factor treatments, PRP offers the benefit
of maintaining the normal physiologic ratios of these
molecules.
The precise relationship between platelet count and
growth factor concentrations remains unclear. Some studies
have shown that platelet count is correlated with the type and
quantity of growth factors released following PRP treatment.
For example, Sundman et al. recently found positive correla-
tions between platelet counts and both TGF-β1 and
PDGF-AB concentrations within PRP preparations [7].
Since TGF-β1 and PDGF-AB are considered to be anabolic
growth factors, the authors suggest that platelets increase
anabolic signaling.
This is consistent with the clinical effects of PRP on
facilitating tendon repair [8, 9]. On the other hand, others
have argued that the simple assumption that platelet con-
centration is directly proportional to growth factor concen-
tration is wrong [10]. Linear regression analysis performed
by Eppley showed little correlation between platelet num-
ber and growth factor concentration, with high variation
from patient to patient [11]. Although they did find signifi-
cant increases in VEGF (6.2×), PDGF-BB (5.1×), EGF
(3.9×) and TGF-β1 (3.6×) in PRP relative to whole blood,
the correlation between platelet count and these growth
factors was low, with the best correlation at only 60.4 % for
TGF-β1 [11]. Zimmerman et al. showed that up to 50 % of
the variation in growth factor concentrations can be
accounted for by the degree of contamination with WBCs
[10]. They also showed that storage conditions such as tem-
perature and time from initial preparation influence the
total growth factor content. A recently published study also
suggested that platelet-derived growth factors can become
trapped in the fibrin glue associated with PRP gel prepara-
tions [12].
4 A.S. Wasterlain et al.

The relationship between platelet count and cellular


response is also ambiguous. A comparison of the effects of
different platelet concentrations on fibroblast expression of
type I collagen levels did not show a dose-response relation-
ship to increasing platelet concentrations [6]. However,
Haynesworth et al. showed that human mesenchymal stem
cell proliferation is proportional to the platelet concentra-
tions in PRP, and begins at a four- to fivefold increase in
platelet numbers [13]. Similarly, Lui et al. showed that increas-
ing platelet concentrations and acidic pH levels stimulate
fibroblast proliferation and type I collagen production [14].
Importantly, although PRP has been shown to enhance
healing from tendon injury, clinical improvement is not nec-
essarily correlated with platelet concentration. For example,
an in vivo animal model of anterior cruciate ligament
healing showed a 24 % decrease in cellular density of repair
tissues treated with 3× relative to 5× platelet concentration,
but no significant differences in structural or mechanical
properties of the ligament [15]. Alternatively, excessively
high platelet concentrations may actually have deleteri-
ous effects on healing [3]. In a study comparing the effects
of PRP with low (two million platelets/μL) and high (five
million platelets/μL) platelet concentrations on intestinal
anastomoses, the lower platelet concentration promoted
anastomotic wound healing, whereas the higher concentra-
tion inhibited healing [16].

Leukocytes

By contrast with platelets, which increase anabolic signaling,


leukocytes contain and produce cytokines that are cataboli-
cally active and promote inflammation (Table 1.1). High
leukocyte count has been associated with higher release of
VEGF [17, 18]. Dragoo et al. have also demonstrated a large
acute inflammatory response with increased cellularity and
vascularity in rabbit tendons 5 days after treatment with
leukocyte-rich (LR-PRP) relative to leukocyte-poor PRP
(LP-PRP) (Fig. 1.1). The same study showed that the acute
inflammation was resolved by 14 days.
Chapter 1. Contents and Formulations 5

Table 1.1 Growth factors associated with different cell types


Cell type Associated growth factors
Leukocytes PDGF, VEGF
Neutrophils MMP-9, IL-1B
Monocytes IL-1B
Platelets PDGF-AA, PDGF-AB, PDGF-BB, VEGF,
TGF-B1

a b

c d

Figure 1.1 Representative high-powered fields (400× magnifica-


tion) are shown for each tendon treatment group at 5 days post-
injection of saline (a), whole blood (b), leukocyte-rich PRP
(LR-PRP) (c), and leukocyte-poor PRP (LP-PRP) (d). Tendons
treated with saline (a) appear relatively normal while those treated
with LP-PRP (d) show a small inflammatory response. Tendons
treated with whole blood (b) show some inflammatory cell infiltra-
tion, while those treated with LP-PRP (c) show marked infiltration
by macrophages and lymphocytes
6 A.S. Wasterlain et al.

Leukocyte concentration is positively associated with


catabolic gene expression and negatively associated with
matrix gene expression in tendon and ligament [19].
Neutrophil granules contain collagenases, gelatinases, lyso-
zymes, elastases, serprocidins and myeloperoxidase, which
facilitate tendon and ligament degradation [20]. Specifically,
neutrophils are associated with greater MMP-9 and IL-1B
concentrations, and monocytes are associated with greater
IL-1B concentrations in PRP [7]. The antagonistic relation-
ship between platelets and leukocytes, and the growth fac-
tors and cytokines they produce, suggests that the ideal
orthopaedic PRP preparation might have a high ratio of
platelets to leukocytes, thereby promoting anabolism over
catabolism.
On the other hand, it is possible that leukocytes could
play an important antimicrobial role in PRP, particularly
intra-operatively, where the risk of infection is greater. For
example, a retrospective analysis of 1400 patients showed
that LR-PRP significantly reduced chest wound infections,
and decreased chest and leg wound drainage after coro-
nary artery bypass surgery [21]. Although LR-PRP reduces
pain and inflammation following open and closed orthope-
dic procedures [22, 23], the effect of LR-PRP has not been
directly compared to that of LP-PRP.

Fibrin

In response to cellular injury in vivo, thrombin cleaves solu-


ble fibrinogen into fibrin monomers that assemble into net-
works of insoluble fibrin polymers. Ultimately, these
networks form a provisional matrix at the site of injury in
which platelets, leukocytes, and other cells can proliferate,
organize, and execute specialized functions [24]. Tissue
regeneration therapies, including platelet-rich preparations,
often aim to replicate and modify these naturally occurring
matrices. Because the structure and function of these fibrin
Chapter 1. Contents and Formulations 7

matrices is determined by several conditions including clot-


ting and polymerization rates [24], it is important to consider
fibrinogen concentration, fibrin density, and polymerization
when evaluating tissue regeneration therapies [25].
Liquid PRP formulations contain soluble fibrinogen, which
is the precursor molecule to fibrin monomers. Fibrinogen
modulates the activity of monocytes and macrophages and
therefore mediates the transition between inflammatory and
regenerative stages of injury response [26]. Fibrinogen is a
heterogeneous molecule, with differences dependent on post-
translational protein modifications as well as genetic variants
[27]. Therefore, the cellular responses to fibrinogen or fibrin
matrices within PRP applications may be highly individual-
ized. Unlike gel or membrane forms of PRP, liquid PRP does
not contain a fibrin matrix ex vivo.
Gel or membrane-like applications contain varying forms
of fibrin matrices that may serve as scaffolds for tissue regen-
eration. However, because all gel or solid matrix applications
of PRP are derived from the initial liquid blood fraction,
fibrin density is determined by the concentration of fibrino-
gen during preparation. Most PRP protocols ultimately pro-
duce a low-density fibrin matrix suitable for surgical
application but lacking the true support network that may aid
healing [28]. Another important factor, fibrin polymerization,
is determined by the ratio of fibrinogen and thrombin and the
final biomechanical properties of the PRP product. High
thrombin concentrations trigger rapid polymerization, lead-
ing to formation of a dense network that hampers cytokine
signaling and cellular migration. Conversely, slow polymer-
ization creates a more flexible scaffold and an environment
that is more conducive to cellular migration and cytokine
signaling [29].
Some commercial PRP formulations may allow prepara-
tion of either liquid or gel PRP, which may be useful for cer-
tain clinical applications (see section “Commercial PRP
Systems” below).
8 A.S. Wasterlain et al.

Table 1.2 Roles of growth factors and other molecules contained in


PRP
Role Pro- Anti-
Angiogenesis Angiopoietin-1, Endostatins,
[40, 41] CD40L, HGF, platelet factor-4,
PDGF, TGF-B1, thrombospondin-1,
VEGF B-thromboglobulin
Inflammation EGF, IL-1B, HGF
PDGF, VEGF
Matrix TGF-B MMP-9
deposition
Cellular EGF, FGF, HGH,
proliferation IGF-1, PDGF,
and migration TGF-B

Growth Factors and Cytokines

PRP contains numerous growth factors, whose properties vary


significantly (Table 1.2). PRP typically contains a three to five-
fold increase in growth factor concentrations. Anabolic activity
and ability to induce chondrocyte and tenocyte differentiation
are important properties to consider in orthopaedics.
Anabolic growth factors contained in PRP include TGF-
β1, PDGF, IGF-1, VEGF, human growth hormone (hGH) and
bFGF. IGF-1 enhances fibroblast proliferation, which is criti-
cal to tendon repair. Since it is produced by the liver, IGF-1
is primarily contained in plasma, and thus is constant in most
PRP preparations regardless of platelet count. VEGF, PDGF
and TGF-β1 are key growth factors derived from platelets.
Both IGF-1 and hGH are positively correlated with musculo-
tendinous collagen expression [30]. VEGF is a powerful
stimulator of angiogenesis, building new vasculature to bring
additional extrinsic cells, nutrients, and growth factors to the
site of injury [31, 32]. TGF-β1 improves collagen synthesis
and deposition in vitro, and regulates cell proliferation, divi-
sion and apoptosis [33–35]. PDGF is primarily found and
stored in the α-granules of platelets [36], and thus is directly
Chapter 1. Contents and Formulations 9

proportional to the number of platelets in PRP. It is chemo-


tactic to macrophages and fibroblasts, enhances fibronectin
and glycosaminoglycan deposition, and increases cell activity
early in the healing response [37, 38]. bFGF, otherwise known
as FGF-2, contributes to angiogenesis by stimulating the pro-
liferation of endothelial cells, and interacts with TGF-β and
PDGF-BB to enhance proliferation of satellite cells, the stem
cells of mature muscle [39].
Catabolic growth factors found in PRP include MMP-9 and
IL-1β. MMP-9 degrades collagen and other extracellular
matrix molecules, and is associated with poor healing. IL-1β is
a potent inflammatory cytokine, and has been implicated in
autoinflammatory diseases, tendinitis and trauma. Injured
human rotator cuff tendons contain increased IL-1β, and IL-1β
triggers its own positive feedback loop, increasing IL-1β fur-
ther and generating a massive inflammatory response [39].
While PRP contains some growth factors that are known
to be anabolic, is also contains many molecules that may
antagonize its effect on tissue metabolism. For example, PRP
contains many growth factors that are pro-chondrogenic,
including TGF-β1, IGF-1, bFGF, and BMP-2. However, most
preparations also contain high levels of anti-chondrogenic
growth factors, such as VEGF, IGFBPs, PDGFs, and
EGF. Additional studies have shown that while chondrogenic
growth factors such as TGF-β1 directly stimulate type I col-
lagen production in skin, synovium and tendon, PRP prepa-
rations containing the same amount of TGF-β1 actually
inhibit collagen production [6]. These results reflect the com-
plex molecular pool contained in PRP, and point to the pos-
sible antagonistic effects of many of these growth factors. This
finding has sparked interest in modifying PRP preparations
to eliminate the unwanted, antagonistic growth factors.

Platelet-Rich Formulations
Two main issues muddy the current PRP literature. First,
there is little consensus regarding the exact constituents of
PRP; the only definition consistently upheld in the literature
10 A.S. Wasterlain et al.

defines PRP as a volume of autologous plasma that has a


platelet concentration above normal baseline, which typically
ranges from 150,000/μl to 350,000/μl [4]. Second, naming sys-
tems used to classify PRP formulations are highly inconsis-
tent. For example, platelet-poor plasma (PPP) gel is also
referred to as fibrin glue or fibrin sealant. Similarly, PRP gel,
PRP fibrin matrix, and PRP membrane are used interchange-
ably by some authors, while others document subtle differ-
ences in preparation protocols or final biomechanical
properties.
However, regardless of nomenclature, all preparation
techniques falling under the PRP umbrella have several
things in common [25]. Blood is first collected from the
patient with anticoagulant and immediately centrifuged
within the hour. This initial centrifugation separates red
blood cells (RBCs) from acellular, platelet-poor plasma
(PPP) and the “buffy coat,” which contains concentrated
platelets and ± white blood cells (WBCs). Through various
other steps, the RBC and PPP layers are discarded and the
platelet concentrate remains. The platelets contained within
this layer may be activated using thrombin, calcium chloride,
or environmental factors, and this layer can then be applied
to the injury site as a liquid injection or as a gel following
additional processing. PRP (including leukocyte-poor PRP),
leukocyte-rich PRP (LR-PRP), and preparation rich in
growth factors (PRGF) are commonly injected liquid
preparations. This type of treatment can be performed in a
minimally invasive, outpatient setting and delivers the acti-
vated preparation directly to the site of injury, resulting in an
immediate release of growth factors that may influence cells
for up to 7 days [42, 43].

Platelet-Rich Plasma (PRP)

Multiple definitions have been proposed for PRP. One defini-


tion is an autologous blood fraction with a platelet concentra-
tion of at least one million platelets/μl, or approximately five
Chapter 1. Contents and Formulations 11

times higher than that of whole blood [4, 44]. Alternatively,


others have proposed any platelet concentration above nor-
mal baseline, which typically ranges from 150,000/μl to
350,000/μl [4]. PRP is derived by drawing a small volume of
blood (usually 25–50 ml), separating the plasma, platelet and
red blood cell components by centrifugation, and extracting
approximately 3–5 ml of the platelet-rich “buffy coat” layer.
This platelet-and growth factor-rich autologous fraction may
then be pre-activated with the clotting factor thrombin or
directly injected back into the patient at the site of muscle or
tendon injury. Within PRP, various preparation methods lead
to the production of leukocyte-rich PRP (LR-PRP),
leukocyte-poor PRP (LP-PRP), or Preparation Rich in
Growth Factors (PRGF).

Leukocyte-Rich PRP (LR-PRP)

Leukocyte-rich PRP includes leukocytes in the autologous


blood fraction. Because a separation system is required in
order to exclude leukocytes, the majority of the protocols
used in the current literature yield LR-PRP.
Several recent studies have characterized the leukocyte
concentration produced by commercially available PRP
preparation systems [7, 45]. These investigations have raised
questions regarding the role of leukocytes in PRP application.
A recent study by Dragoo et al. evaluated the inflammatory
effect of intratendinous injection of LR-PRP compared with
LP-PRP, whole blood, and saline [46]. Tendons injected with
LR-PRP showed a heightened acute inflammatory response
at 5 days compared with other treatment groups. The remain-
ing available literature is limited, but previous studies have
shown mixed results, some reporting that concentrated deliv-
ery of leukocytes to a site of injury may hinder the healing
response or amplify the release of catabolic, pro-inflammatory
mediators [19, 47, 48], while others contend that leukocytes
may play an important antimicrobial role and enhance
growth factor release [10]. Commercially available systems
12 A.S. Wasterlain et al.

include: Arteriocyte/Medtronic Magellan (Cleveland, OH),


Biomet GPS III (Warsaw, IN), EmCyte GenesisCS (Fort
Myers, FL), Harvest SmartPReP 2 (Plymouth, MA).

Leukocyte-Poor PRP (LP-PRP)

Leukocyte-poor PRP specifically excludes leukocytes in the


autologous blood fraction through the use of cell separator
systems. Compared with LR-PRP, LP-PRP does not elicit a
large inflammatory response at 5 days post-intratendinous
injection [46]. At 14 days post-injection, tendons treated with
LR-PRP and LP-PRP showed similar inflammatory reponses.
One particular type of LP-PRP known as preparation rich in
growth factors (PRGF) appears frequently in existing litera-
ture. PRGF, also known as Endoret (endogenous regenerative
technology), refers to a specific protocol developed by Anitua
et al. [49] and often refers to the use of the PRGF System II
(BTI, Vitoria-Gasteiz, Spain). This protocol ultimately yields
LP-PRP that can be converted into a fibrin matrix or mem-
brane [50]. Commercially available LP-PRP systems include:
MTF/CONMED Cascade (Edison, NJ), Cytomedix Angel
(Gaithersburg, MD), BTI PRGF-Endoret (BTI, Vitoria-
Gastiez, Spain), EmCyte Pure PRP (Fort Myers, FL), Cascade/
Aesthetic Factors SELPHYL (Bethlehem, PA), and Arthrex
ACP (Naples, FL).

Platelet-Poor Plasma (PPP)

Platelet-poor plasma is a byproduct of the PRP preparation


process and is the blood fraction devoid of platelets that is
produced following centrifugation to separate red blood cells
from the plasma. Some protocols recommend double-spin
centrifugation to ensure that platelets are pelleted and con-
tained within the PRP layer. PPP does not have the same
therapeutic advantages because it lacks the platelet-derived
growth factors and cytokines. However, it still contains the
Chapter 1. Contents and Formulations 13

full complement of plasma proteins responsible for the


coagulation cascade and can be used clinically to aid hemo-
stasis. A 2007 porcine study found that PPP improved wound
healing when compared with untreated controls, but was not
as effective as PRP [51]. Many commercially available PRP
kits also produce a platelet-poor plasma fraction (Table 1.3).
In addition to serving as a direct mechanism of growth fac-
tor delivery, derivatives of PRP can also be used as a tissue
scaffold. These types of applications such as gels and fibrin
matrices allow for a longer, more prolonged release of growth
factors and provide a more mature fibrin network. There are
slight variations in the viscosity, elasticity, suturability, and
plasticity of this class of biomaterials. Accordingly, some gels
are injectable preparations, while many fibrin matrices and
membranes must be directly applied to the injury site, often
via surgical intervention. Additionally, specific technical char-
acteristics of some kits may affect the amount and kinetics of
the release of platelet-derived growth factors [52].

PRP Gel

PRP gel is a broader category encompassing platelet-rich


fibrin matrix and platelet-rich fibrin membrane applications.
These preparations include fibrin, and are designed to pro-
vide some structural support for the tissue repair process.
PRP gels can be produced ex vivo from any liquid PRP for-
mulation by the addition of thrombin and calcium chloride,
which initiates fibrin polymerization. Accordingly, PRP gel
may include or exclude leuckocytes, depending on the cellu-
lar content of the original liquid fraction. Including a more
mature fibrin matrix may be advantageous for some platelet-
rich applications. Fibrin matrix formation is part of the natu-
ral wound healing response, and has been shown to enhance
delivery of platelet growth factors [53]. Many commercially
available PRP kits also contain an activator that can be used
to produce a PRP gel (Table 1.3). Commercially available
systems marketed specifically as PRP gels include: Cytomedix
Table 1.3 Comparison of mean cell counts among major commercial PRP preparation systems
14

Blood Enriched Increase in Platelet Increase in Mishra


volume volume Gel [Platelets] capture [WBCs] class
required produced Processing PPP activator (times efficiency (times PAW
System Company (mL) (mL) time (min) produced? available? baseline) (% yield) baseline) class
Leukocyte-Rich PRP

GenesisCS EmCyte 60a 4–5 [73] 12–16a [73] + 10a 68–96 %a 5a P4-Aα 1A
[74]

GPS III Biomet 60a 6a 27 [73] + + 2.1–8.1 [7, 23–90 %a 4.3–5 [7, P2-Aα 1A/B,
A.S. Wasterlain et al.

Clotalyst 45, 68] [45, 67] 45] or 2A/B

Magellan Arteriocyte/ 60 [72] 1–10 [73] 17–22 [72, + 2.8–7.1a 66 % [45] 2–2.9 [45, P2-Aα 1A/B
Medtronic 73] [45, 72] 72]

SmartPReP 2 Harvest 60 [72] 10 [73] 15–17a [72] + 6–8.2a [72] 62–72 % 3.5–4a [72] P2-Aα 1A
[65, 67,
69]

Leukocyte-Poor PRP

ACP Arthrex 11–16a 4a 5 + 2.1a 60 %a 0.13 [7] P2-Bβ 3B or


4B

Angel Cytomedix 40–180a 1–20a + 6–8a 3A

Cascade MTF/ 18 [45] 7.5 [45] 21 [73] + 1.6 [45] 68 % [45] 0.2 [45] P2-Bβ 3B or
CONMED 4B
PRGF- BTI 9a 2–4 [75] 28a + 2–3a P2-Bβ 3B or
Endoret 4B

Pure PRP EmCyte 50a 6a 9.0a 2.2a 3A

SELPHYL Cascade/ 20 [73] + 1.3 [65] 66 % [65] 0.03 [65] 3B or


(FIBRINET) Aesthetic 4B
Factors

PRP gel

AutoloGel Cytomedix + 78 %a

Clotalyst Biomet 11a 4–10a 20a + + 2A/B

Plateltex ACT Plateltex + + 4.4 [51]


a a a a
RegenKit Stryker/ 9 4–5 10 1.7a 95 %a 2–6×a 2B
RegenLab
Vivostat PRF Vivostat 120a 5–6a 25a + 3.8–7a [65] 17 % [65] 0.04 [65] 2A/B
a
Data obtained from manufacturers’ promotional literature
Abbreviations: ACP Autologous Conditioned Plasma, BTI Biotechnology Institute, LP-PRP leukocyte-poor platelet-
rich plasma, MTF Musculoskeletal Transplant Foundation, PCCS Platelet Concentrate Collection System, PPP
Chapter 1. Contents and Formulations

platelet-poor plasma, PRGF plasma rich in growth factors, PRP platelet-rich plasma, LR-PRP leukocyte-rich platelet-
rich plasma
15
16 A.S. Wasterlain et al.

AutoloGel (Gaithersburg, MD), Biomet Clotalyst (Warsaw,


IN), Plateltex ACT (Bratslava, Slovakia), RegenLab/Stryker
RegenKit (Le Mont-Sur-Lausanne, Switzerland), and Vivostat
PRF (Medicon Valley, Scandinavia).

PPP Gel (Fibrin Glue and Sealant)

This group comprises platelet-poor plasma gel, also known as


fibrin glue or fibrin sealant, which is produced by adding cal-
cium chloride to the PPP blood fraction. Because PPP still
contains all of the clotting proteins, it forms a fibrin matrix
when activated with calcium chloride. It has been shown that
the fibrin matrix alone may enhance healing by providing a
conductive scaffold for cell migration and new matrix forma-
tion [3, 28]. Currently, there are no commercially available
systems aimed specifically at providing PPP gel. However, the
Plateltex ACT (Bratislava, Slovakia) system produces PPP,
and also includes gel activation reagents.

Activation Procedures
The term “activation” refers to two key processes within PRP
preparations that may be initiated: (1) degranulation of plate-
lets to release α-granules containing growth factors and (2)
fibrinogen cleavage to initiate matrix formation. The bulk of
current literature simply indicates the means by which gen-
eral PRP activation occurs but rarely specifies which cellular
components are targeted by these techniques. The addition of
thrombin and calcium chloride, or collagen are both effective
in activating both platelets and fibrinogen, while activation
via freeze/thaw cycles initiates degranulation only.
Accordingly, rapid platelet activation can be achieved by the
following three mechanisms: (1) addition of calcium chloride
and thrombin, (2) freeze/thaw cycles, and (3) direct exposure
to collagen in vivo. Once activated, the PRP composition is
often referred to as the PRP releasate.
Chapter 1. Contents and Formulations 17

Calcium Chloride and Thrombin Activation

In the calcium chloride (CaCl2) and thrombin activation


approach, thrombin directly activates platelets via a proteo-
lytic G-coupled protein receptor. Calcium then replenishes
the binding site previously bound by anticoagulant.
Extracellular proteolysis results in platelet aggregation and
intracellular α-granule lysis [54]. Although different protocols
exist, the standard clinical approach uses a 10:1 ratio of
thrombin and CaCl2 (142.8 U/ml thrombin, and 14.3 mg/ml
CaCl2D2H2O) [43, 55].
Thrombin activation leads to very rapid, irreversible plate-
let activation and degranulation, releasing growth factors
such as VEGF, PDGF-BB and TGF-β [55, 56]. Higher con-
centrations of calcium and thrombin may also lead to higher
extracellular concentrations of PDGF-BB and TGF-B. A
disadvantage of calcium chloride and thrombin activation is
the requirement of using an exogenous substance, thus
increasing the risk of infections and allergic or other reac-
tions. This is especially problematic with the use of bovine
thrombin, which is immunogenic.
Alternatively, either thrombin or calcium can be used
independently to stimulate platelet activation. Calcium-
mediated activation simultaneously initiates growth factor
release and polymerization of fibrinogen into fibrin [57].

Freeze/Thaw Cycle Activation

Freeze/thaw cycles are used for in vitro PRP applications.


PRP is stable for 5 days at room temperature [58–60] and for
extended periods when frozen. Subsequent thawing lyses
platelets and releases contents from platelet α-granules [61].
The goal of freeze/thaw activation is to physically injure the
platelets to initiate de-granulation. However, there is no
consensus regarding the precise number of freeze/thaw
18 A.S. Wasterlain et al.

cycles necessary for complete degranulation. Many protocols


suggest that four freeze/thaw cycles are adequate for in vitro
studies.
This method is useful for in vitro laboratory experiments
because it utilizes a physical, not chemical, mechanism of
platelet activation. Consequently, it does not require the
addition of any substrates, and thus does not alter the con-
tents of the sample. However, this method is time-intensive
and thus is impractical for clinical applications.

In Vivo Collagen Activation

Finally, non-activated PRP can be injected directly into the


injured tissue, which becomes activated upon contact with
collagen. Collagen is one of the most potent activators of
platelet adhesion and aggregation [62]. Specifically, thrombo-
genic fibrillar collagen types I and III are the most potent
platelet activators, given their high content of von Willebrand
Factor, an important substrate that mediates the interaction
between collagen and platelets [62].
In vivo collagen activation is the preferred method for
activating PRP in many clinical applications, because it
leads to a slower and more sustained release of growth fac-
tors relative to the thrombin method. Collagen activation
leads to more sustained release of TGF-B1, and an 80 %
greater cumulative release over a 7-day period relative to
thrombin activation [56, 63]. On the other hand, cumulative
VEGF release appears unaffected by activation method,
and PDGF has shown mixed results. Clots formed using
type I collagen had significantly less retraction than those
formed using bovine thrombin [63]. Additional benefits of
in vivo collagen activation are that it can be injected
through a smaller gauge needle because the clot has not yet
formed, and that it eliminates the risk of immunologic reac-
tions to exogenous activating substances such as calcium or
thrombin.
Chapter 1. Contents and Formulations 19

Commercial PRP Systems


At least 16 commercial platelet separation systems are avail-
able today, many of which may vary significantly in the rela-
tive amounts of platelets, leukocytes, erythrocytes, and
anabolic and catabolic growth factors. This has two important
implications. First, it is difficult to generalize results from
clinical trials using a given PRP manufacturer to anticipate
clinical outcomes using a different commercial PRP prepara-
tion. On the other hand, physicians may be able to tailor their
choice of PRP system to the patient’s unique needs.
Although in vitro studies have begun to elucidate the cel-
lular and molecular contents of different commercial PRP
systems, it remains unclear how these differences affect clini-
cal outcomes in patients. Commonly reported platelet param-
eters include platelet concentration and increase from
baseline in whole blood, and platelet recovery or capture
efficiency. Multiple investigations have assessed the compo-
nents of various commercially available platelet-rich
preparation systems, the results of which are summarized in
Table 1.3.

PRP Classification Systems


Many researchers in the field have emphasized that not all
PRP is created equal. As outlined in Table 1.3 and described
above, PRP kits vary widely in their platelet, leukocyte, and
erythrocyte counts, as well as in their delivery methods. These
differences make it difficult to compare the results of basic
science and clinical research studies, and may also have a
meaningful effect on the clinical utility of specific PRP for-
mulations. In recognition of this issue, a few PRP classifica-
tion systems have been proposed.
DeLong et al. devised the “PAW” system, which refers to
the absolute Platelet count, platelet Activation method, and
the presence or absence of White blood cells [71]. Platelet
count is categorized as ≤ baseline (P1), baseline to 150,000
20 A.S. Wasterlain et al.

(P2), 750,000 to 1,250,000 (P3) or >1,250,000 (P4). Activation


method is categorized as exogenous (x) or not (no letter
assigned). White blood cells are categorized as above baseline
(A) or below baseline (B), and neutrophil count is also cate-
gorized as above (α) or below (β) baseline. The authors point
out that ultimately the researcher or clinician decides whether
to activate the PRP endogenously via direct contact with col-
lagen or exogenously, regardless of the commercial kit used.
Mishra et al. have also proposed a classification system
that accounts for the leukocyte and platelet counts, as well as
activation method [72]. They defined four PRP types: leuko-
cyte-rich (WBCs increased over baseline) non-activated (1),
leukocyte-rich activated (2), leukocyte-poor (minimal or no
WBCs) non-activated (3), and leukocyte-poor activated (4).
Sub-types are assigned based on platelet content: >5× plate-
lets (A) or <5× platelets (B).

Effect of PRP on the Cellular Environment


The cellular environment in which platelet-rich preparations
are placed strongly influences the direction and magnitude of
tissue response. In orthopaedics, the majority of PRP research
has focused on its effects on connective tissues such as ten-
don, ligament and muscle. Principal components of the ten-
don extracellular matrix include collagen (65–80 % dry
weight), elastin (1–2 %), and ground substance. Tendons
contain primarily type I collagen, which confers strength.
Elastin provides flexibility and elasticity. The key cellular
constituents of these tissues are discussed here.

Fibroblasts

Fibroblasts are critical to maintaining structural integrity of


connective tissues, and they initiate the earliest molecular
events leading to tissue repair. Anitua et al. demonstrated
that skin, synovium and tendon fibroblast proliferation is
stimulated by growth factor-rich preparations [6]. However,
Chapter 1. Contents and Formulations 21

only synovial cells showed a dose-dependent proliferative


response to platelet concentration. In addition, tendon fibro-
blasts responded significantly more than synovium or skin
fibroblasts to pro-angiogenic signals in plasma preparations.

Collagen

PRP triggers differentiation of tendon stem cells into active,


proliferating tenocytes capable of producing high quantities
of collagen, which contributes to the repair of injured ten-
dons with cell and matrix deficits [64]. The primary form of
collagen in healthy tendons is type I collagen (COL1).
Injured tendons have a higher percentage of type III (COL3),
which has fewer cross-links between and within tropocolla-
gen units. One in vitro study showed that platelet-poor and
platelet-rich clot releasates decreased expression of both
COL1 and COL3 in human tendon cells, with no significant
change in the relative ratio of collagen types [39]. On the
other hand, another study demonstrated an increase in COL3
but not COL1 gene expression by human anterior cruciate
ligament (ACL) cells after exposure to PRP, and attributed
the increase in total collagen production in PRP-treated cells
to cellular proliferation, rather than to increased production
of collagen by existing cells [65].

Tenocytes

PRP also triggers differentiation of tendon stem cells into


active, proliferating tenocytes capable of producing high
quantities of collagen, which contributes to the repair of
injured tendons with cell and matrix deficits [56].

Systemic Effects

Please refer to Chapter 9 for a complete discussion of the


systemic effects of PRP.
22 A.S. Wasterlain et al.

Conclusion

Platelet-rich preparations may serve an important role in


accelerating the tissue repair and regeneration processes of
the cellular response to injury. The potential of these platelet-
rich preparations is largely due to the growth factors and
cytokines derived from platelets and the presence of clotting
proteins that aid in the formation of early matrices. The cel-
lular composition, final biomechanical properties, and cellu-
lar environment into which the treatment is placed are
important considerations when evaluating platelet-rich
applications.

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Chapter 1. Contents and Formulations 29

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245–51.
Chapter 2
Platelet-Rich Plasma in Pain
Medicine
José Fábio Santos Duarte Lana, Eduardo Fonseca Vicente,
Adam Weglein, William Dias Belangero, Fabrício Dias Assis,
and André Marques Mansano

Summary
For the past 20 years, autologous Platelet-Rich Plasma (PRP)
has been safely employed and its use has been documented in
many areas, including orthopedics, sports medicine, dentistry,

J.F.S.D. Lana () • E.F. Vicente


The Bone and Cartilage Institute – IOC,
Av. Presidente Kennedy, 1.386, 2º andar, salas 26, 28 e 29,
Indaiatuba SP CEP 13334-170, Brazil
e-mail: josefabiolana@gmail.com
A. Weglein, DO, DABMA
University of Texas Houston Medical School, Houston, TX, USA
University of North Texas Health Science Center,
Ft Worth Texas 6800 West Loop South Ste 500, Bellaire,
TX 77401, USA
W.D. Belangero
Department of Orthopedics and Traumatology – Faculty of Medical
Sciences, University of Campinas,
Tessália Vieira de Camargo 126, Campinas, SP CEP 13083-852,
Brazil
F.D. Assis • A.M. Mansano
Interventional Pain Practice-WIP – Singular – Pain-Control Center,
R. Maria Monteiro 968, Campinas, SP CEP 13025-151, Brazil
N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 31
Practice, DOI 10.1007/978-1-4471-7271-0_2,
© Springer-Verlag London 2016
32 J.F.S.D. Lana et al.

neurosurgery, ophthalmology, urology, wound healing, cosmet-


ics, cardiothoracic, otorhinolaryngology and maxillofacial
surgery.
In the past few years, scientific research and technology
have afforded fresh perspectives for understanding the
wound healing process. Initially, platelets were used solely to
assist in clotting. However, platelets are also responsible for
releasing many bioactive proteins and growth factors respon-
sible for recruitment of macrophages, mesenchymal stem
cells, and osteoblasts, which not only promotes necrotic tissue
removal, but also improves the quality of tissue regeneration
and the healing process.
Based on this principle, platelets are now used in clinical
practice to stimulate the supraphysiological release of cells
responsible for healing, so as to increase healing potential,
mediate inflammatory processes and reduce pain through the
release of substances such as serotonin, histamine and dopa-
mine. PRP has become an important prophylactic alternative
in pain medicine and in treatment of chronic injury.

Introduction
Platelet-rich plasma (PRP) is an autologous biomaterial
obtained by centrifuging whole blood. PRP may be defined
as a fraction of autologous plasma with platelet concentration
above baseline level. Studies have shown that ideal concen-
tration is at least a fourfold increase in initial concentration,
or around 1,000,000 mm3 [1].
Platelets are responsible for hemostasis promotion, new
connective tissue formation and revascularization. A sample
of whole blood usually contains 93 % red blood cells, 6 %
platelets and 1 % white cells (leukocytes).
Justification for the benefits of PRP rests on inverting the
proportion of these cells in the blood, reducing the red layer
to 5 %, since red blood cells are less useful in the healing
process, and increasing platelets and leukocytes to 94 % to
stimulate tissue regeneration [1].
2 Platelet-Rich Plasma in Pain Medicine 33

Other benefits that justify PRP use are the diverse growth
factors contained in the concentrate, such as transforming
growth factor-beta (TGF-β), platelet-derived growth factor
(PDGF), basic fibroblast growth factor (bFGF), insulin-like
growth factor-l (IGF-l), vascular endothelial growth factor
(VEGF), and epidermal growth factor (EGF) [2–4]. There
have been in vitro and in vivo reports of the effects of PRP
and growth factors on the stimulation of fibroblast prolifera-
tion by IGF-l, bFGF, and PDGF and of collagen synthesis and
extracellular matrix synthesis by TGF-β in tendons and liga-
ments [5–8], resulting in enhanced regeneration and increased
tissue strength and consistency.

PRP Processing
PRP processing involves the separation and concentration of
platelets, leukocytes, and growth factors, considered initiators
in any repair process. However, there is no standard tech-
nique for obtaining PRP, and different preparations are
described in the literature. There is wide variability in the
ability to concentrate these cells and, for the most part, this
variability relies on equipment, manufacturer and materials
utilized.
Several authors have presented their proposals for classi-
fying and suggestions for standardizing the infinite number of
terms observed in current studies. These classifications are
directly related to the cell composition of each concentrate.
Mishra [9] classifies PRP on the basis of the presence or
absence of leukocytes, on the utilization of activating agents
and on final platelet concentration, enabling a framework
wherein products are divided into four types: type 1, high
leukocyte concentration, non-activated; type 2, high leuko-
cyte concentration, activated; type 3, non-activated, no leuko-
cytes or low leukocyte concentration; and type 4, activated, no
leukocytes or low leukocyte concentration. All these product
types may be further classified as A, corresponding to platelet
concentration equal to or five times higher than the baseline
34 J.F.S.D. Lana et al.

level, or B, platelet concentration five times lower than base-


line concentration.
Everts et al. (2008) [10] based their terms on two underly-
ing principles: (1) the impossibility of obtaining a leukocyte-
free product and (2) the need for platelet activation which
generates a gelled product, internal or externally. Thence they
proposed the term “platelet and leukocyte gel” (PLG) to
denominate any and all platelet-derived products, assuming
the veracity of premises 1 and 2.
Anitua et al. (2009) [11] advocate production of a
leukocyte-free platelet concentrate which, according to the
authors, can promote proinflammatory effects because of the
proteases and acid hydrolases contained within. The authors
suggest use of the term PRGF (plasma rich in growth fac-
tors), hinged upon the principle that activation of any platelet
concentrate will result in the release of growth factors, essen-
tial agents in the healing cascade.
Ehrenfest et al. (2010) [12] uphold that it is impossible to
obtain a leukocyte-free platelet concentrate, and suggest clas-
sification based on the presence or absence of leukocytes and
on fibrin network architecture. This classification allowed to
allocate the different products into four categories: pure
platelet-enriched plasma (P-PRP), which includes PRGF
proposed by Anitua et al. (1999) [13] and leukocyte- and
platelet-rich plasma (L-PRP), pure platelet-rich fibrin
(P-PRF), leukocyte- and platelet-rich fibrin (L-PRF).
Another much-debated and researched point is the use of
the leukocyte layer in association with the platelet concen-
trate. Some studies have shown that white blood cells con-
tained in PRP naturally safeguard this substance from
infectious and allergic processes [14, 15].
However, there is still much debate surrounding leukocyte
use, and studies suggest that, when leukocytes are present,
neutrophils are able to release metalloproteinases which cause
cell matrix degradation as well as release free radicals [16]. This
could lead to a delayed healing response from muscles [17].
According to Sundman et al. (2006), growth factor concen-
trations and catabolic cytokines are influenced by PRP cell
2 Platelet-Rich Plasma in Pain Medicine 35

composition. Platelets augment anabolic signaling whereas


leukocytes increase catabolic signaling molecules, thereby
suggesting reduction or non-utilization of the leukocytes con-
tained in platelet concentrates [18].
Ehrenfest et al. (2012) also studied platelet concentrates,
linking fibrin network and leukocyte content to capacity and
speed of release of some growth factors. The leukocyte-dense
concentrate was responsible for the slower and more intense
release of growth factors, mainly, TGFβ1. The researchers
concluded that polymerization and final architecture of the
fibrin network strongly influence intensity and growth factor
release speed, mainly TGFβ1, and that leukocyte presence
plays an essential role in forming this network [19].
Platelet concentrates containing leukocytes may addition-
ally be classified as different types. They include neutrophils,
monocytes/macrophages and lymphocytes. They play differ-
ent roles in tissue healing.
Neutrophils are phagocytes and contain more than 40
hydrolytic enzymes. Their activation leads to phagocytosis of
debris and release of free oxygen radicals and proteases. This
release of toxic molecules from neutrophils may be condu-
cive to secondary muscle damage [17, 20]. It is not yet known
what effects neutrophils have on soft tissue injury, whether
acute or chronic.
Macrophages are the tissue form of circulating monocytes
and their job is debris clearance, mainly phagocytic in nature.
They also have a role in reflections concerning the pro-
inflammatory and anti-inflammatory aspects of healing [21].
Since it is only possible to fractionate different kinds of white
cells within PRP, it is likely that the absence of macrophages
is more unfavorable to healing than any neutrophil-induced
damage.
PRP and PH activation of the concentrate are some other
parameters currently being discussed. Bovine thrombin, col-
lagen, autologous thrombin and calcium have been utilized to
activate platelets previously activated by anti-coagulants.
Many current clinicians are not activating PRP, and achieve
equal clinical results.
36 J.F.S.D. Lana et al.

This combination results in the formation of a gel, which


can be utilized in open surgery and in wounds, but cannot be
injected, even with a large caliber needle. Bovine thrombin,
collagen and calcium activation produce intense PRP activa-
tion and consequently the rapid release of platelet growth
factors. This event is still being discussed because it is not yet
known whether it is ideal to promote such activation and
consequent early release of growth factors. Ehrenfest et al.
(2012) [19], concluded from an in vitro study that, if PRP is
activated intensely with calcium or bovine thrombin, the
fibrin network will be unstable. If PRP is activated in a more
physiological manner, a stable, tetramolecular network forms.
Because of this, autologous thrombin use intended to pro-
mote a more physiological environment is being increasingly
encouraged.
When considering the actual PRP procedure, we have to
address the use of co-administered analgesics, as isolated
PRP can be quite painful when injected into target tissues.
Most clinicians employ some sort of anesthetic, i.e. local
block with lidocaine or direct mixing of the PRP product with
anesthetic. New research, pending publication, suggests a
possible negative effect of combining high dose anesthetics,
i.e. lidocaine with PRP. This negative anesthetic effect is dose
dependent: lower doses are considered as cytotoxic at
present.

Injection Technique

Ultrasound-Guided Peripheral Blocks

The use of ultrasound to guide peripheral nerve blocks has


grown more frequent. Some neurostimulation-related bene-
fits of this technique have been demonstrated in the literature.
The leading ones are: lower failure rate, shorter procedure
time, shorter latency time and longer duration of block with
lower risk of accidental vascular puncturing [22–27].
2 Platelet-Rich Plasma in Pain Medicine 37

Common injections are extremely inaccurate when done


“blind”, with failure rates for shoulder joint injections around
73 % [28], and failure rates for hip joint injections estimated
at around 20–40 % [29].
Some authors believe that image guidance is absolutely
crucial to ensure adequate intra-articular needle placement.
Accuracy of palpation-guided needles is highly variable (50–
93 %) in various different techniques. Moreover, 100 % accu-
racy was demonstrated in ultrasound-guided intra-articular
injections into cadaver knees, performed by professionals
with limited experience, versus 55 % in palpation-guided
injections. These data support the benefits of injecting bioma-
terial to the exact site of the condition, whether in a ligament,
tendon or muscle.
With its lower probability of causing vascular injury, ultra-
sound has become an interesting tool for peripheral block
guidance. Association of the two techniques, ultrasound and
neurostimulation, though it may make the procedure more
costly, enhances safety and further reduces the risk of failure
[30, 31].
Today, with the advances in ultrasound equipment and
methods, it is possible to identify vascular and neural
structures highly accurately. Therefore, ultrasound-guided
injections enable greater accuracy, both in reaching the spot
to be treated and in minimizing the risk of accidental vascular
injury. Success and complication rates are related to the clini-
cian’s experience, as is the case with any other technique [32].

Ultrasound-Guided Anesthetic Blocks


for Differential Pain Diagnoses
Ultrasound-guided anesthetic nerve blocks, using local anes-
thetic, associated or not with corticosteroids, opioids and
other agents, may be performed for the purposes of diagno-
ses, prognoses and/or therapy. Ultrasound-guided anesthetic
blocks relieve pain by interrupting the sensitive pathways
38 J.F.S.D. Lana et al.

that carry information to the central nervous system. They


may be used in association to other kinds of anesthetic
blocks, such as trigger point injections, joint injections or inte-
grated to treatments that may involve, according to disease
stage, other therapeutic measures for the same patient, such
as systemic medication (painkillers, anti-inflammatory drugs,
muscle relaxants, opioids) and physical methods (physical
therapy, hydrotherapy, RPG, Pilates) [33].
Ultrasound-guided anesthetic blocks are included in treat-
ments of interdisciplinary nature. They are indicated in keep-
ing not only with pain characteristics, but also with patient
profile with respect to acceptance of such method [34].
Ultrasound-guided anesthetic blocks for diagnostic pur-
poses may be employed to determine the anatomical source
of pain and to guide differential diagnosis of peripheral or
central, local or referred and visceral or somatic pains. With
relation to chronic pain, after undergoing several imaging,
laboratory and functional tests, it is common for patients, to
remain doubtful about the cause of their pain. In this case,
when we block the sensory pathway with local anesthetic,
promoting a temporary interruption of the pain, we are able
to define the anatomical source of pain and, thus, define the
cause of the pain, enabling us to better direct the course of
treatment [35–37] (Figs. 2.1, 2.2, 2.3 and 2.4).
Diagnostic and therapeutic interventional techniques have
developed greatly in the past few years and, together with
pharmacological and non-pharmacological treatment, consti-
tute one of the mainstays of chronic pain treatment. The
efficacy of interventional techniques in pain treatment
depends on the correct placement of needles and other
devices in known nerve structures, guided by well-established
anatomical landmarks [38, 39].
Intra-articular knee injections are generally performed by
orthopedic surgeons and rheumatologists and, with the
expanding role of physicians in chronic pain management,
common injections are frequently done in primary care set-
tings. This trend highlights the need for process standardiza-
tion to ensure patient safety and comfort by employing the
most accurate injection techniques possible [40–42].
2 Platelet-Rich Plasma in Pain Medicine 39

Fig. 2.1 Hip. Drawing of “pain map” based on VAS – (Visual


Analogue Scale) – Ultrasound-guided diagnostic anesthetic block

Fig. 2.2 Knee. Drawing of “pain map” based on VAS – (Visual


Analogue Scale) – Ultrasound-guided diagnostic anesthetic block

A number of imaging methods may be used to help the


physician identify the correct path for the diagnostic and/or
therapeutic intra-articular injection, including those guided
by radiograph, ultrasound, computer tomograph and mag-
netic resonance. However, ultrasound presents one of the
most practical options because it is safe, fast, relatively low-
cost, and does not emit radiation [43].
Though a number of studies have reported intra-articular
accuracy of common injections using images or anatomical
landmark guidance, few controlled studies comparing the
accuracy of these methods have been carried out. Results
from available analyses show that utilization of imaging
guidance improves the accuracy of intra-articular injections
into large joints, including the knee. Furthermore, ultrasound
guidance, specifically in the knee, greatly increases the prob-
ability of correct needle placement [43].
Accurate ultrasound-guided intra-articular injections have
led to positive results, and preliminary clinical evidence sug-
gest that these patient benefits may result in reduction of
health costs in the long term [44].
40 J.F.S.D. Lana et al.

Fig. 2.3 Diagnostic anesthetic block to Achilles tendon

Fig. 2.4 Diagnostic anesthetic block to supraspinatus tendon

In the U.S.A., only one in five rheumatologists regularly


utilize ultrasound in their musculoskeletal practice, even
though three out of four agree that it should be a standard
clinical tool, for diagnosis, injection guidance, and evaluation
of treatment response [44].
2 Platelet-Rich Plasma in Pain Medicine 41

PRP in Orthopedics and Pain Medicine


In pain medicine, the primary goal is pain relief and increase
in functional capacity of the injured tissue. Therefore, treat-
ment should not only target local pain reduction, but also
provide an environment where healing is stimulated, promot-
ing tissue regeneration.
Several authors have reported successful use of Platelet-
Rich Plasma (PRP) therapy in treating musculoskeletal inju-
ries, with positive and promising results in pain reduction and
injury healing. Several studies describing PRP use have
emerged in which there were no complications.
There have been frequent reports of significant reduction
in pain, risk of amputation, decrease in bleeding and conse-
quent improvement of quality of life among patients.
The analgesic effect of PRP in the post-operative period
was observed by Gardner et al. [45] in patients who had
undergone total knee arthroplasty, who had lower pain inten-
sity and who needed a smaller amount of painkillers. The
same was observed in the post-operative period immediately
after dental surgeries [46]. In addition to the growth factors
located in the alpha-granules, platelets contain other sub-
stances stored in the dense granules, such as serotonin, hista-
mine and dopamine, which possibly have an analgesic effect
and justify these results [47].
Civinini et al. (2011) [48] reviewed in vivo and in vitro
clinical studies in which PRP was used. They concluded that
it was a safe, easily preparable and relatively low-cost proce-
dure for treatment of subacute and chronic injury.
Studies suggest that PRP has beneficial effects on post-
operative inflammation, blood loss, infections, medication
reduction, osteogenesis promotion, and soft tissue healing.
In addition to the hemostasis provided at the vascular injury
site, platelets contain a large amount of growth factors and
cytokines that are fundamental in healing bone and soft tissue
[49]. This leads to greater awareness regarding the role plate-
lets play in the healing process, and has also led to their thera-
peutic application [50].
42 J.F.S.D. Lana et al.

Autologous PRP injections are being used and docu-


mented in many areas, including orthopedics, sports medi-
cine, dentistry, neurosurgery, cardiology, ophthalmology,
urology and injury healing, as well as in the fields of cosmet-
ics, cardiothoracic surgery and maxillofacial surgery, with
positive and promising results.
With the growing benefits being provided in these areas,
PRP gradually started to be studied and utilized in several
branches of orthopedic surgery, mainly because it perfects
and accelerates healing [51].

Tendons and Ligaments


Several studies have supported PRP use in the treatment of
tendinopathies and ligament injuries.
Tendons and ligaments have slower healing potential com-
pared to the majority of tissues because of their sparse vascu-
larization. In a wound healing process, this may result in new
tissue which does not possess the same structural and func-
tional properties as those of the original tissue. One possible
explanation is that their sparse vascularization results in an
inadequate release of growth factors and healing cells at the
site of injury. That is why it is possible that PRP, a source of
growth factors and healing cells, can improve the speed and
quality of healing in these tissues.
Sánchez et al. (2007) [52] evaluated the recovery of 12
athletes who had undergone surgical repair for total rupture
of the Achilles tendon, 6 of them jointly with PRP therapy.
Results showed a significant difference in recovery between
patients treated with PRP and the control group, such as
shorter time for recovery of range of movement, significant
pain relief and shorter time before resuming training.
In a case report, Maniscalco et al. (2008) [53] described the
use of PRP in its gel form for treatment of rotator cuff injury
as an alternative to tendon transposition. The researchers
observed complete wound healing after 6 months of
treatment.
2 Platelet-Rich Plasma in Pain Medicine 43

Knoet et al. (2009) [54] used three separate PRP injections,


at 15-day intervals, to treat patellar tendinopathy, and showed
a significant improvement in physical pain relief, improve-
ment in function and quality of life. Patients were followed up
for 6 months.
In 2010, de Vos et al. [55] evaluated the efficacy of PRP in
the treatment of chronic tendinopathy of the calcaneus ten-
don. In a random, double-blinded, prospective study, the
researchers compared 27 patients who received PRP injec-
tions with 27 patients who received saline injections. The
groups were equally treated with eccentric exercises. No sta-
tistically significant differences were observed between
groups relative to pain and activity. In the study conclusion,
the researchers did not recommend PRP for treatment of
chronic tendinopathy of the calcaneus tendon.
The first randomized clinical study on PRP use in com-
plete calcaneus tendon ruptures was published in 2011 by
Schepull et al. [56]. The results evaluated were elasticity after
7 weeks and functional assessments after 1 year. No differ-
ence was observed between groups relative to elasticity. One
potential detrimental effect of PRP was observed in relation
to functional results after 1 year. It should be noted that in
this study they utilized average platelet concentration, about
10 times the amount found in peripheral blood. Compared to
similar studies, this amount is much higher.
In a recent controlled, randomized, prospective clinical
trial, Almeida et al. (2012) [57] selected 27 patients who
were later divided at random into groups to receive (n –
12) and not to receive (n – 15) PRP injections to the har-
vesting site of patellar tendon during ACL reconstruction
surgery. The results were evaluated by magnetic resonance
imaging (MRI) of the patellar tendon after 6 months. The
researchers observed that the recovery of the patellar ten-
don tear zone/cleft was significantly better in the PRP
group than in the control group. The Visual Analogue Scale
(VAS) was also used and post-operative pain scores were
significantly lower in the group that received PRP. In con-
clusion, they confirmed that PRP could improve tissue
44 J.F.S.D. Lana et al.

healing at the patellar tendon harvesting site. PRP also


reduced post-operative pain.
Several studies have highlighted the beneficial effects of
PRP on ligament injury. There have been frequent reports of
reduction in post-operative pain and decrease in length of
time before resuming previously compromised activities,
such as that of Sánchez et col (2003) who reported fewer
complications and better healing after PRP injections in 100
patients who had undergone ACL reconstruction, in a retro-
spective study [58].
In a randomized, prospective study, Vogrin et al. (2010)
[59] evaluated the use of platelet gel and leukocytes for
reconstruction of the anterior cruciate ligament with tendon
graft in 25 patients, having observed significant improvement
in antero-posterior knee stability in patients treated with the
gel, as compared to the control group. Also, they observed
significant reduction in post-operative pain.
Radice et al. (2010) [60] evaluated data from a prospective,
non-randomized, blind assessment study regarding PRP use in
ACL reconstruction. Fifty patients who had undergone ACL
reconstruction with autologous graft of the patellar or hamstring
tendons were included in this study, and divided into two groups,
in which PRP had been utilized or not in the graft. The evalua-
tion included a series of magnetic resonance tests, done between
3 months and 1 year after surgery, and researchers evaluated
graft homogeneity. In the group that received PRP, the graft
became homogeneous within a period of time 48 % shorter than
that recorded for the control group.

Muscle

Muscle injury often leads to pain and significant impairment,


and is frequently a cause of disability among athletes and
non-athletes. Even though there are many treatment options,
pain and the long duration of current treatments have under-
pinned the search for new therapies.
The basic science of muscle healing has therefore focused
on the use of autologous biological products as alternative
2 Platelet-Rich Plasma in Pain Medicine 45

treatment for muscle injury. The role of diverse growth fac-


tors in natural muscle repair is evident, and is based on the
increase of cytokine levels found in the muscle tissue healing
process. PRP contains many of these bioactive proteins and
growth factors, leading to their utilization also in this kind of
injury.
Despite the importance of these injuries, few clinical stud-
ies evaluate treatment options. Conventional treatments seek
to reduce injury-associated bleeding and swelling. The admin-
istration of anti-inflammatory drugs may relieve pain, none-
theless, there is evidence that these medications interfere
with the healing capacity of muscle tissue. Anti-inflammatory
drugs may inhibit precursor myogenic cell fusion, thereby
hampering muscle healing [61].
Sánchez et al. (2005) evaluated the clinical benefits of
ultrasound-guided injections of growth factors in 20 patients
with sports-related muscle injury [62]. Results demonstrated
reduction in pain and swelling, total recovery of functional
capacity before the expected time and muscle tissue regen-
eration, observed by ultrasound. There was no evidence of
fibrosis in any of the cases treated and there was no recur-
rence of injury among all athletes after they resumed their
normal sports activities.
In a review of the literature, Mishra et al. (2009) [63] evalu-
ated studies on PRP injections to muscles and tendons. The
authors postulate that when PRP is injected in its inactive
form, it is activated by conjunctive tissue collagen. PRP then
releases its growth factors and cytokines. These bioactive pro-
teins, on their part, stimulate local stem cells and increase
expression of the extracellular matrix gene. From this point,
repair cells from bone marrow or local circulation are recruited.
At the same time, PRP inhibits excess inflammation, apoptosis,
and metalloproteinase activity. Interaction between these
pathways results in tissue repair of the muscle, which allows it
to endure effort or sporting activities, thus reducing the pain.
There are few publications involving PRP injections for
treatment of muscle injury. Up to the moment, there are no
publications of randomized clinical studies involving PRP
injection for muscle injury treatment.
46 J.F.S.D. Lana et al.

Hamid et al. (2012) [64] published a protocol where a


blinded, controlled, randomized study will be undertaken.
Twenty eight patients, 18 years and over, with recent Grade II
ischiotibial muscle injury will be invited to participate.
Participants will be distributed at random either to receive
autologous PRP injection and take part in a rehabilitation
program, or only to take part in a rehabilitation program.
Participants will be followed up on the third day post injec-
tion and afterwards on a weekly basis for 16 weeks. At each
follow-up visit, participants will be evaluated with respect to
their capacity to resume playing, using a set of criteria. The
primary endpoint is when participants have met the criteria
for return to their game or at the end of 16 weeks. The main
outcome of this study will be the time taken to resume play-
ing after injury. This study protocol proposes rigorous assess-
ment and with significant potential for utilization for Class 2
muscle injuries. If PRP efficacy is proven, such findings may
greatly benefit patients with similar injuries.

Cartilage
The social impact of degenerative diseases such as joint carti-
lage conditions is increasing, and it is a consequence of the rise
in the average age of the active population [65, 66]. Joint car-
tilage injuries have limited potential for repair, they are diffi-
cult to treat and remain a problem for doctors and orthopedic
surgeons. Capacity for cartilage regeneration is limited
because of its isolation from systemic regulation and because
of vascular deficiency [67–69]. Unlike the majority of tissues,
in an inflammatory process, chondrocytes do not migrate to
the joint injury from a healthy, intact site [41, 43]. Biomechanical,
metabolic, and biological changes, as well as trauma and iso-
lated chondral injuries, may lead to loss of tissue homeostasis,
resulting in faster degeneration of joint surfaces, leading to
cartilage injury or osteoarthritis (OA). These pathologies are
generally known to be crippling diseases, for they are con-
stantly associated to severe pain and mobility difficulties.
2 Platelet-Rich Plasma in Pain Medicine 47

Numerous growth factors are present in joint cartilage.


They work together to regulate the development and mainte-
nance of joint cartilage homeostasis throughout life [70]. In
this manner, PRP has been frequently proposed as a promis-
ing treatment for cartilage regeneration. Various studies
assessing PRP use for treatment of knee osteoarthritis [71–74]
have reported promising clinical results with pain reduction,
functional improvement, speedier return to daily and sporting
activities and consequent improvement of quality of life.
A case report was published in 2003 by Sánchez et al.,
describing PRP use to treat cartilage avulsion. In the patient
treated, 2 ml activated PRP was injected into the space
between the fragment and its bone bed. Treatment was con-
sidered successful, with the patient evolving without pain and
resuming sporting activities 18 weeks after PRP injection.
The authors concluded that the addition of PRP strength-
ened cartilage healing, given the normal prognosis [13].
The positive and promising results of PRP treatment led
Sánchez et al. (2008) to carry out a cross-sectional, observa-
tional study in which 30 patients with knee pain received PRP
intra-articular injections so they could be compared to a
group that received hyaluronic acid (HA) [72]. The patients
received three one weekly injection of PRP or of HA for
three consecutive weeks, and were followed up over a
6-month period. According to the authors, the PRP injection
boosted the success rate by 33.4 %, in comparison with a 10 %
success rate in the HA group. Furthermore, PRP significantly
improved the percentage of physical pain relief in the assess-
ment questionnaires utilized.
Kon et al. (2010) [75] treated 100 patients (115 knees) with
four PRP injections over a 21 day period, and followed these
patients for 12 months. Of the patients evaluated in this study,
58 suffered from degenerative chondral injury with early OA,
and 24 patients were diagnosed with advanced-stage OA.
A significant improvement in functional questionnaires and
visual analogue scale (VAS) scores was observed. 80 % of
patients were satisfied with the treatment, while older
patients had a smaller response in comparison with younger
48 J.F.S.D. Lana et al.

patients. Patients with advanced-stage OA showed significant


improvement in only 30 % of the cases. PRP therapy is safe
and efficient for pain relief, improvement in function and
quality of life in patients with degenerative joint disease.
Sampson et al. (2010) [74] also reported diminished symp-
toms after three PRP injections for treatment of knee OA. In
a prospective, preliminary study, 14 patients with primary and
secondary knee osteoarthritis received three PRP injections
into the affected knee over a period of 4 weeks. To measure
cartilage height, specific pain assessment and functional
capacity questionnaires were utilized, as well as musculoskel-
etal echographies. The study showed significant and almost
linear improvement in knee injuries, including pain reduction
and symptom relief. Most patients reported favorable results
after 12 months from the treatment. The positive trends and
the safety profile that PRP demonstrates could be utilized in
larger blinded randomised studies, to determine whether
PRP is truly efficient in knee osteoarthritis treatment.
Recently, in a study published by Sánchez et al. (2012) [76],
40 patients suffering from severe unilateral hip OA received
three PRP injections, which were administered once a week.
The primary endpoint was significant pain relief, described as
a reduction in pain intensity by at least 30 % from baseline,
assessed by the WOMAC sub-scale at least 6 months after
treatment. The Visual Analogue Scale (VAS) and the Harris
sub-scale for hip pain, were also utilized to verify results.
Secondary outcomes included at least 30 % improvement in
pain and incapacity. Statistically significant reductions in
grading on pain and functional capacity questionnaires were
reported. 57.5 % of the patients reported clinically relevant
pain reduction. The study supports the safety and tolerability
of PRP injections for pain relief and functional improvement
in patients with hip OA.
The basic science, the pre-clinical studies and the clinical
studies collectively indicate that PRP is a promising treat-
ment for cartilage injury and joint pain. Even though the
mechanism of action of PRP has still not been completely
2 Platelet-Rich Plasma in Pain Medicine 49

elucidated, several studies suggest that there is an anabolic


effect on chondrocytes, synoviocytes, with a significant
increase in cell proliferation and matrix production, as well as
an anti-inflammatory effect by means of the regulation of the
known catabolic signalling pathway.

Spine
Spinal pain includes all painful conditions originating
from the spine, whether from intervertebral disks, mus-
cles, ligaments, joints or bones. The structures responsible
for spinal pain are the vertebrae themselves, interverte-
bral discs, spinal cord, nerve roots, facet joints, muscles
and ligaments 1-10. It is a public health problem in
Western industrialized societies. Prevalence rates range
from 12 to 35 % and around 10 % of these patients
become chronically disabled. In the U.S.A., 1.5–4 million
adults suffer from lumbar pain secondary to spinal degen-
eration for whom conservative treatment was ineffective,
and many of these patients eventually undergo surgical
procedures [77].
Back pain is closely related to intervertebral disk degen-
eration (IDD). This condition, though asymptomatic in
many cases, is also associated with sciatica and interverte-
bral disk herniations or prolapses. Studies have shown that
discogenic pain is the most common cause of chronic back
pain, with rates ranging from 30 to 60 % in all cases. IDD
alters disk height and the resting mechanisms of the spinal
column, possibly adversely affecting the behavior of other
spinal structures, such as muscles and ligaments. In the long
run, it may lead to spinal stenosis, an important cause of
pain and disability in the elderly; its incidence is increasing
exponentially with current demographic changes and
increase in life expectancy.
Disks degenerate earlier than any other musculoskeletal
tissue structures. There is a significant reduction in end-plate
50 J.F.S.D. Lana et al.

vascularization before the end of the first decade of life which


marks the beginning of a structural disorganization of the
intervertebral disc. Around the age of 20, the end-plate and
the subchondral bone severs blood supply to the interverte-
bral disc [78, 79] and, after that, the intervertebral disc begins
to depend on diffusion for nutrients [80]. About 20 % of
people in their teenage years have discs with degenerative
signs and this number can reach 60 % of severe disk degen-
eration around the age of 70.
Current treatments only attempt to reduce the pain, rather
than repair the degenerated disc. Therefore, these approaches
are only palliative. The more conservative treatments are rest
(not recommended), muscle relaxants, corticosteroid or local
anesthetic injections and manipulation therapies. Several
minimally invasive interventions are also used, such as
chemonucleolysis, intradiscal radiofrequency, annuloplasties
and percutaneous decompression. Although some trials
report successful outcomes, these interventional therapies
have been effective in about 50 % of patients, and still consti-
tute palliative treatments. Surgical treatment of IDD must
always remain the last option because of the related compli-
cations and also because they do not provide a cure. Disk
prostheses and arthrodeses are the most common.
Disk degeneration can lead to changes in adjacent tissue
and is a risk factor for development of spinal stenosis in the
long term. Biological therapies aim to restore disk height
and its biomechanical function, therefore, they will not only
serve as palliative treatment, but they will also change the
natural history of the disease. Many studies have demon-
strated the benefits of some biological therapies for the
treatment of degenerative disk disease, such as those dis-
cussed below.

Glucosamine and Chondroitin

These agents have been used in several peripheral joint


osteoarthritis studies. There is evidence that glucosamine and
2 Platelet-Rich Plasma in Pain Medicine 51

chondroitin synergistically increase the natural response of


chondrocytic hypermetabolic repair and delay cartilaginous
enzymatic degradation. Derby et al. conducted a pilot study
utilizing intradiscal injections of glucosamine and chondroi-
tin with DMSO (dimethyl sulfoxide) and hypertonic dextrose
to promote a repair response in the intervertebral disc [81].
Clinical efficacy was similar to IDET procedures but more
cost-effective. There is a need for comparative, controlled,
randomized studies to establish the efficacy of intradiscal
glucosamine and chondroitin injections.

Cell Therapies

The goal of cell therapies is to promote matrix regeneration


in degenerated intervertebral disks. The disk cells can be
negatively influenced by an increase in load (pressure), by
hypoxia and nutrient deprivation. In response, they secrete
lactate, cytokines and proteases that will induce acidification
and disk degeneration. This degenerated matrix can cause
end-plate sclerosis, sensitize nociceptors and exacerbate the
adverse effects of load and decreased transport. Growth fac-
tors can increase intracellular disk matrix synthesis up to
fivefold.
In 1991, Thompson et al. demonstrated strong effects of
TGF-β 1, a growth factor, on disk cell proliferation and on
proteoglycan synthesis [82]. In 2000, Gruber et al. showed
that platelet-derived growth factors have an antiapoptotic
effect on fibrous annulus cells [83], besides stimulating annu-
lar cell proliferation after 4 days of exposure [84]. In turn,
PRP contains high TGF-β 1 concentrations and platelet-
derived growth factors [3, 4].
In a study with rabbits, Obata et al. showed an increase in
the number of chondrocytes in the nucleus pulposus after
intra-discal PRP injection. Radiological and histological
management also demonstrated reparative effects on the
degenerated disk, with an increase in disk height in compared
to the control group [85]. Several other studies in animals
52 J.F.S.D. Lana et al.

have found promising results using growth factors to reduce


disk degeneration [86–90].
In a trial in patients who had undergone anterior spinal
fusion, Hartmann et al. demonstrated increased bone density
in the fusion area in the group that had received PRP intra-
operatively in comparison with the control group [91].
Levi et al. demonstrated good results of intradiscal plate-
let-rich plasma in a small cohort of discogenic pain patients.
The main limitation of this study was the non-controlled
methodology and the lack of discography as a diagnostic tool
in many patients [92]. Tuakli-Wosornu et al. randomized 47
patients with discogenic pain selected by provocative discog-
raphy and showed greater results in the patients submitted
do intradiscal platelet-rich plasma injection compared with
the placebo group in 8 weeks follow-up [93]. Once the
medium and long term PRP effects is well established, it will
be not surprising if even greater results be achieved in a lon-
ger follow up.
The use of cell therapy for spinal degenerative disease
represents a great hope in the treatment of this complex con-
dition. Randomized studies in humans should be conducted
so that this therapeutic possibility can be evaluated.

Conclusion
Platelet-rich Plasma emerges as an autologous, non-
immunogenic, therapeutic option capable of stimulating the
supraphysiological release of cells responsible for wound
healing, with the aim of augmenting healing potential, medi-
ating inflammatory processes and relieving pain. The fre-
quently reported reduction in pain and in healing time
renders the PRP technique an important therapeutic tool in
interventional pain treatment. Correct association with
adjunctive therapies is also related to a good pain prognosis.
It is important to remember that being an autologous bioma-
terial, the success of PRP therapy depends on the patient’s
general clinical conditions. The present literature reports
2 Platelet-Rich Plasma in Pain Medicine 53

mixed results on the effects of PRP [94]. Therefore, careful


assessment of the mechanical and biological factors involved
in the wound healing process must be performed so as to
focus treatment and guide precise indication of the tech-
nique. Assuredly, all these effects related to the use of growth
factors will continue spurring research into the ideal role of
PRP and its main indications in pain medicine and
orthopedics.

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2 Platelet-Rich Plasma in Pain Medicine 61

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Chapter 3
PRP: Tips for Application
in the Musculoskeletal
System
Steven Sampson, Ken Mautner, Alessio Giai Via,
and Angie Botto-van Bemden

Introduction
Common chronic musculoskeletal injuries, including tendi-
nopathies, cartilage disorders, and spine disease are difficult
to manage. Their treatments are often only palliative, aiming
to reduce pain. Traditional interventions include non-steroi-
dal anti-inflammatory medications, corticosteroid injections,

S. Sampson, DO ()
The Orthohealing Center and The Orthobiologic Institute (TOBI),
David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
e-mail: drsampson@orthohealing.com
K. Mautner, MD
Rehabilitation Medicine, Orthopaedics and Sports Medicine,
Emory University, Atlanta, GA, USA
A.G. Via, MD
Department of Orthopaedic and Traumatology, University of
Rome “Tor Vergata”, School of Medicine, Viale Oxford 81,
Rome 00133, Italy
A.B.-v. Bemden, PhD, ATC, CSCS
Musculoskeletal Research International (MRI), Clinical Research
Experts (CRE), Florida International University, Miami, FL, USA

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 63


Practice, DOI 10.1007/978-1-4471-7271-0_3,
© Springer-Verlag London 2016
64 S. Sampson et al.

ice, rest, and bracing or immobilization. Unfortunately, most


of these therapeutic options are limited in scope and target
symptoms rather than the underlying disease. Along with
new insights regarding tendon, cartilage, and intervertebral
disc pathophysiology, a promising new era of biologically
based cellular therapy has emerged. Orthobiologics involves
the inclusion of biology and biochemistry in the develop-
ment of bone and soft tissue replacement materials for skel-
etal and tissue healing [1]. Platelet Rich Plasma (PRP)
therapy is the first readily available autologous “bedside”
orthobiologic injectable gaining widespread use.
PRP has been defined as the supernatant obtained follow-
ing low G-force centrifugation of a unit of whole blood, which
produces an increase over baseline platelet count [2]. PRP has
been used widely not only in sports medicine, but in multiple
fields including wound care, trauma surgery, ophthalmology,
cosmetic surgery, maxillofacial surgery, and others [3]. There is
great variability and a lack of standardization regarding the
optimal mode of platelet preparation. Differing cellular com-
ponents in manual or commercially prepared PRP may or
may not include leukocytes and red blood cells (RBC), and
have exogenous activation requirements. A classification sys-
tem has been proposed to optimally interpret varying study
protocols (Fig. 3.1) [4]. Additionally, the authors have recently
submitted a revised classification to further clarify whether or
not RBC are present in the PRP (Fig. 3.2). Despite widespread
use, no standardization of PRP preparation, injection

White Blood Cells Activation Platelet Concentration

Type 1 Increased No Activation A = 5x or > B = < 5x

Type 2 Increased Activated A = 5x or > B = < 5x

Type 3 Minimal or No WBCs No Activation A = 5x or > B = < 5x

Type 4 Minimal or No WBCs Activated A = 5x or > B = < 5x

Fig. 3.1 PRP Classification (Reprinted with permission from Mishra


et al. 2011)
3 PRP: Tips for Application in the Musculoskeletal 65

WBC’s Activation? Platelet RBC’s


Concentration
Type 1 Increased No activation A. 5x or > R+. Increased
B. <5x R-. Minimal to
no RBC’s
Type 2 Increased Activation A. 5x or > R+. Increased
B. <5x R-. Minimal to
no RBC’s
Type 3 Minimal to no No activation A. 5x or > R+. Increased
WBC’s B. <5x R-. Minimal to
no RBC’s
Type 4 Minimal to no Activation A. 5x or > R+. Increased
WBC’s B. <5x R-. Minimal to
no RBC’s

Fig. 3.2 Revised PLRA PRP Classification System

technique, or post treatment rehabilitation has been estab-


lished. This chapter will give an overview of the current appli-
cations of PRP technology, with expert hints and tips following
the authors’ clinical experience in the use of PRP in the man-
agement of musculoskeletal disorders.

PRP Preparation

Leukocyte Rich vs. Leukocyte Poor PRP

Many PRP formulations are available for clinical uses, yet the
products may vary greatly, and this may influence the efficacy
of treatment and the results of clinical trials. The presence of
WBCs is largely dependent on the centrifugation and tech-
nique used to process the PRP. The role of leukocytes in PRP
(L-PRP) is controversial [5]. Those in favor of excluding WBCs
argue that neutrophils may have a detrimental effect on muscle
and bone [6, 7]. Furthermore, neutrophils can release degrada-
tive matrix metalloproteinase (MMP)-3,8,9 and 13, and free
radicals [6]. This could lead to a delayed healing response in
muscle [7]. Regarding bone healing, mice with temporary neu-
tropenia and femur fractures healed the fracture with a higher
bending moment of the bone callus compared to the non-neu-
tropenic mice [8].
66 S. Sampson et al.

In support of using L-PRP, leukocytes seem to have an


important role in tissue healing. Discussing the isolation of
specific cell lines, it is important to consider the loss of natu-
ral physiologic interplay of leukocytes with each other, espe-
cially monocytes, stem cells and macrophages [9].
Concentrated monocytes and platelets exert an anti-
inflammatory control on neutrophils [3, 10]. Much of the
healing seen with L-PRP is related to a combination of its
growth factors and the anti-inflammatory, including RANTES
production, the blocking of MCP-1 release from monocytes,
and the concentration of LXA4 [10]. Mc Carrell et al. [11]
demonstrated, using tendon-like cells harvested and cultured
from horse flexor digitorum superficialis tendons, that an
increase in leukocyte content of PRP products is positively
correlated with an increased expression of inflammatory
cytokines and that the platelet/leukocyte ratio had no influ-
ence on this effect. Leukocytes also release stem cell chemo-
tactic agents including VEGF. These recruited stem cells have
far reaching healing potential for ligaments, tendons and
bone. Over the past 10 years, the authors used a wide range
of table -top centrifuges (MTF Cascade, Arthrex ACP,
Arteriocyte Magellan, Harvest Smart PReP2, Cytomedix
Angel, Biomet GPS II and III, Emcyte, and manual prepara-
tions) which produce both L-PRP and Leukocyte poor PRP
(Fig. 3.3). Some of these systems include RBC while others
do not. The current thoughts of the authors, based primarily
on their clinical experience, are in support of limiting concen-
trations of RBC and WBC (especially neutrophils), in an
intra-articular environment, while chronic tendinopathies
may benefit from L-PRP. The positive clinical results in sup-
port of this may at least partially result from the stimulation
and migration of stem cells to the target tissue along with the
complex interplay of WBCs in tissue healing [5].

PRP With or Without RBCs

RBCs may be deleterious when using PRP in soft tissue and/


or joint applications. Traditionally, buffy coat PRP systems
with higher platelet concentrations also have high residual
3 PRP: Tips for Application in the Musculoskeletal 67

Fig. 3.3 Emcyte PRP system

RBCs, 5–15 %. Autologous conditioned platelets (ACP),


plasma based systems, and Platelet Lysate generally carries
minimal to no RBCs. More recently, several commercial PRP
machines that are buffy coat systems are now filtering out the
RBCs, making a PRP with higher platelet counts, and little to
no RBCs and WBCs.
There are some historical and emerging data that indicate
that removing RBCs from PRP is beneficial to treating joint
and possibly soft tissue pathology. Foremost, RBCs have a
negative effect on chondrocytes and in vivo and in vitro stud-
ies have shown that recurrent hemarthrosis, classically associ-
ated with hemophilia, has been associated with arthritic
changes in the knee joint [12, 13]. This cartilage damage has
been demonstrated with even a single exposure to RBCs as
might be obtained from a sports related knee injury [14].
The only direct evidence examining RBCs effects on mes-
enchymal stem cells (MSCs) production was obtained by
Centeno et al. [15]. Comparing RBC-PRP to a pure PRP
68 S. Sampson et al.

preparation in an in vitro investigation, they demonstrated, in


samples collected from patients with ages ranging from 20’s
to 40’s, dramatically reduced colony forming units in the
RBC-PRP compared to one without RBCs. At present, sev-
eral of the leading clinicians in the field of regenerative medi-
cine are filtering out RBCs in their PRP preparation without
much clinical evidence to state whether they obtain better
outcomes. Given these emerging data, the authors are limit-
ing, whenever possible, injecting RBCs into joints and have
begun using RBC poor PRP for soft tissue application.

Platelet Activation and PRP Anti-coagulation

The idea of activating the PRP product is based on the need


to start the clotting cascade to release the growth factors
(GFs) contained in the platelets. There are 4 ways in which
platelets are naturally activated in vivo: the Adenosine
diphosphate (ADP) pathway; the membrane phospholipids-
arachidonic acid system; direct exposure to collagen; and
induction by thrombin [16]. In the clinical setting, calcium
chloride, a cofactor for converting prothrombin to thrombin,
will activate the platelets. Autologous or bovine thrombin can
also be used to rapidly activate the platelets and to produce
a fibrin-like membrane. Bovine thrombin used to activate
PRP has been associated with life threatening coagulopathies
as a result of antibodies to clotting factors V, XI, and throm-
bin. However, since 1997, production has eliminated contami-
nation of bovine thrombin with bovine factor Va. Prior to
1997, Va levels were 50 mg/mL; now, they are 0.2 mg/mL, with
no further reports of complications [17].
To maintain a purely autologous injectable and reduce
adverse reactions promoting a more gradual release of GFs,
the authors do not use bovine thrombin. However, in surgical
applications including rotator cuff tears or hip labral tears, a
gel matrix using autologous thrombin or CaCl with PPP
(Platelet Poor Plasma) may be desired. Despite ex vivo labo-
ratory evidence arguing that activation results in greater
growth factor release, the authors have not seen any advantage
3 PRP: Tips for Application in the Musculoskeletal 69

to this, and therefore generally prefer a sustained release of


GFs without activation. When placing PRP in tendons, liga-
ments and muscles, calcium chloride is not needed since the
platelets will likely activate gradually upon direct contact
with collagen fibers, which is a natural activator in these
tissues.
Also, there are many variables in centrifuge protocols for
different PRP systems. Some of these factors may be related
to the increased risk of mechanically-induced premature acti-
vation. For example, the speed at which the blood spins when
considering the fragility of the cells, and whether a breaking
mechanism exists are just two of the variables to consider.
Some PRP kits include anti-coagulants to prevent initiation
of the clotting cascade, hence minimizing the risk of early
activation. However, once an anticoagulant is added, the pH
of the PRP solution may be altered. Subsequently, this may
influence the normal physiological action of released growth
factors.
Additionally, the use of local anesthetics to prevent pain
when injecting PRP could compromise its therapeutic poten-
tial. Therefore, careful use of local anesthetics or limiting
their use is recommended [18].

Current Applications

Tendinopathy

While histological examination of synovium in osteoarthritic


joints supports the presence of inflammatory cytokines in the
synovium in painful osteoarthritis, the same has not been
shown in chronic tendinopathies [19]. In tendinopathies,
examination of the painful structures yields a distinct absence
of inflammatory markers while also demonstrating the clear
presence of disorganised collagen, neovessels, and fibrosis
[20, 21] (Fig. 3.4). With limited results from treatments aimed
at halting inflammation, the roles of regenerative therapies
are being regarded with great interest. Injecting a high con-
70 S. Sampson et al.

Fig. 3.4 Common Extensor Tendinosis with Neovascularization

centration of platelets into areas of chronically injured ten-


don should release multiple growth factors through the
α-granules of a platelet. These GFs, when present in higher
than physiologic concentrations, assist the body’s own healing
response [22, 23]. Platelets also release additional bioactive
proteins such as stromal-derived factor 1α, which attract mes-
enchymal stem cells, macrophages, and fibroblasts [24, 25].
These bioactive proteins enhance tissue regeneration and
healing, and remove degenerated and necrotic tissue [26, 27].
Basic science investigations show an adequate number of
laboratory and animal studies that reveal increased endoge-
nous growth factors, improved tendon proliferation, and
enhanced collagen deposition [28]. There is growing clinical
research in humans supporting the use of PRP in chronic
non-healing tendinopathies, most notably tennis elbow [29].
Mishra and Pavelko [30] showed positive results in a case
series for resistant tennis elbow, and favorable outcomes
have also been reported in the treatment of plantar fasciitis
[31]. Peerbooms et al. demonstrated a significant improve-
ment in treating lateral epicondylopathy with PRP vs. steroid
3 PRP: Tips for Application in the Musculoskeletal 71

injection: PRP demonstrated superiority up to 1 year post


treatment, with results remaining stable by the 2 year mark
[32]. There is a negligible difference in treatment effective-
ness between PRP, cortisone and saline injections at 12
weeks post-treatment [33]. With this technique, however, a
high volume of lidocaine (10–15 mL) was injected to the
paratenon prior to treatment which could adversely affect
the outcome. This treatment protocol is different from the
previous published investigations [30, 34]. There were also
significant differences in outcome when the PRP patients
were compared to the steroid patients at 6 months and 1 year
in favor of significantly greater pain reduction in the PRP
group (P = 0.005 and P = 0.006, respectively). This paper high-
lights that the clinical effects of PRP for tendinopathy are
delayed, and may not occur until 3–6 months post
treatment.
Often, PRP is not recommended as a first line treatment
except in elite athletes or patients with extensive tissue dam-
age hoping to avoid surgery. Additionally, patients need man-
aged expectations as this treatment is by no means a panacea,
and generally takes several weeks for improvement to be
noted. In fact, typically patients experience temporary worse
pain from the procedure for 5–7 days. Rarely, patients may
have a prolonged inflammation or “flare” following an injec-
tion, the causes of which are unknown. In persistent post
injection pain, range of motion and rehabilitation are critical
to accelerate healing.
In contrast, a subset of patients return to function expedi-
tiously: these patients are “rapid responders.” A placebo
response might contribute to this phenomenon, or, alterna-
tively, these patients may be particularly sensitive to the
serotonin released from the dense granules of platelets, alter-
ing the subjective experience of pain. Further research is
warranted to better understand the subjective and objective
symptomatic inflammatory response following treatment and
the underlying role of hormonal and nutritional status, medi-
cal history, and psychological components.
72 S. Sampson et al.

In many cases, patients will require only one PRP injection


treatment. In scenarios of severe partial tendon tears, calcific
tendinopathies, or chronic resistant tendon injuries, 1–3 PRP
treatments may be needed to sustain long term clinical ben-
efit. Typically, patients are followed up at 8 weeks following
initial injection to determine the need for further treatments.
At that point however, it is frequently too premature to
visualize sonographic improvements of tendon and ligament
which usually requires several weeks to months.

Ligament Pathology

The authors first began treating medial collateral ligament


tears in elite professional soccer/football players 12 years ago
with very positive outcomes. In general, our patients experi-
enced recovery times and return to play 50 % faster than the
expected timeframe with prompt intervention. This elicited
further exploration and application into other regions includ-
ing tendon and cartilage.
Most ligamentous evidence is derived from studies focus-
ing on anterior cruciate ligament (ACL) reconstruction.
Sánchez et al. [35] investigated whether the application of a
particular PRP preparation rich in growth factors (PRGF)
gives a potential advantage for better tendon graft ligamen-
tization after ACL reconstruction. The study involved 37
volunteers who underwent ACL reconstruction with an
autologous hamstring tendon with or without PRGF, and
who required second-look arthroscopy for different reasons.
During the second-look arthroscopy biopsy specimens were
harvested uniformly from the grafted tendons. PRGF treat-
ment influenced the histologic characteristics of the tendon
graft, resulting in tissue that was statistically significantly
more mature than in controls. The histologic samples in the
study group showed newly formed connective tissue envelop-
ing the graft in 77.3 % of patients compared to 40 % of con-
trols. Furthermore, the remodeling of PRGF-treated grafts
involved the formation of synovial-like tissue enveloping
3 PRP: Tips for Application in the Musculoskeletal 73

the graft that conferred a similar appearance to the normal


ACL. PRGF influenced the histological characteristics of
tendon grafts, resulting in better remodeling compared with
untreated grafts. Although the findings are not conclusive, the
use of PRP was safe, and improved pain and graft remodel-
ling [36, 37]. However, more research is needed to study the
effects of PRP treatment on ligament reconstruction and
healing.

Muscle Pathology

PRP was thought to induce muscle scarring. However, more


recent trends in PRP research suggest facilitation of healing
in muscle strains. Higher level of pain relief and quicker
recovery in acute muscle tears with PRP injections have
been reported [38]. Further research is required to clarify
the possible role of PRP for acute and chronic muscle
strains, and its impact on return to sport. Typically, a post
traumatic hematoma mimics whole blood, and contains
approximately 94 % RBC, 4 % platelets, and <1 % leuko-
cytes. Thus, replacing the plug clot with PRP limits the
amounts of RBCs present at the injury site, and increases
growth factor (GF) concentration from platelets (Fig. 3.5).
Proposed mechanisms include increased satellite cell acti-
vation, increased diameter of regenerating fibers, and stim-
ulation of myogenesis. However, recent well conducted
level 1 evidence studies showed no benefit of intramuscular
PRP compared with placebo injections in patients with
acute hamstring injuries in the time to return to play, re-
injury rate, and no benefit of a single PRP injection over
intensive rehabilitation in athletes [39, 40]. However, both
studies had inherent limitations: a blind (without hands on
sonographic guidance) palpation injection technique was
used, and low platelet concentrate or autologous plasma
concentrate (ACP) were injected. Currently, there is little
clinical evidence to support the use of PRP in skeletal
muscle injuries [41].
74 S. Sampson et al.

Fig. 3.5 Ultrasound Guided Aspiration of Quadriceps Hematoma

Intervertebral Disc and Spine Pathology

To date, research for PRP use in spine pathology is limited,


with most studies focusing on intra-operative PRP adminis-
tration during spinal surgery. Several publications use animal
models, but all human studies are limited to in vitro bioengi-
neered disc tissue derived from mesenchymal stem cells. All
studies thus far relating to non-operative spine PRP adminis-
tration are restricted to PRP injections into disc tissue for
discogenic pathology (induced degenerative disc disease or
annular tears). Although PRP is well established in its ability
to cause proliferation, controversy remains as to whether it
has sufficient capability to trigger differentiation of the new
cells into the proper phenotypic expression of intervertebral
disc cells, which are predominantly chondrogenic [42–44].
3 PRP: Tips for Application in the Musculoskeletal 75

There are no published studies related to PRP injection into


soft tissue of the spine, such as spinal ligaments, bony attach-
ments of spine musculature onto vertebrae, and facets joints/
capsules. The authors have found positive trends in PRP with
its application to these spine structures under ultrasound
guidance and/or fluoroscopic guidance. In regards to facet
mediated axial spine pain, positive and negative predictors
are being identified based on treatment outcome. Positive
indicators of successful facet PRP treatment include isolated
facet mediated pain with imaging evidence of facet arthropa-
thy, history of whiplash injury (typically cervical spine), tem-
porary significant relief with chiropractic adjustments, and
positive diagnostic intra-articular injections with lidocaine
and cortisone. Negative predictors include coinciding moder-
ate to severe degenerative disc disease, history of spine sur-
gery, and greater than grade 2 spondylolisthesis. Of the
patients who underwent PRP facet injections in our clinic,
65–70 % had at least a 50 % pain reduction and did not prog-
ress to facet medial branch radiofrequency.
Cellular differentiation is crucial in achieving structural
repair, including new disc cells with the ultimate goal of pain
reduction. Cytokines that promote inflammation, such as
Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-
α), are associated with degenerated intervertebral discs
(IVD) [44]. These catabolic markers serve as targets for
potential treatments to repair the degenerative disc and
restore the normal healthy disc milieu. Differentiation is
proposed to be paramount in repairing degenerative discs
and restoring normal healthy disc tissue. Ideally, the PRP-
induced shift from degeneration to regeneration will clini-
cally manifest as less pain, while disc integrity continues to
improve for months after PRP. However, the concept of pain
in regards to discogenic processes is not well understood,
with some patients showing imaging evidence of severe
degenerative disc processes yet never experiencing
symptoms.
Animal and in vitro studies have shown a strong chon-
drogenic quality of PRP in the spine. Nagae et al. used a
76 S. Sampson et al.

rabbit model to study PRP application in degenerated discs,


and injected it via a gelatin hydrogel into the nucleus pulpo-
sis of induced degenerative intervertebral discs. Histological
evidence revealed new proteoglycans in the disc cellular
nuclei of the treatment group [45]. In 2009, Chen et al.
found up-regulation of chondrogenesis evidenced by
increased markers in an in vivo porcine organ culture sys-
tem which was consistent with the same increased genetic
expression in ex vivo degenerative intervertebral disc sys-
tem derived from mesenchymal stem cells [46]. Chen et al.
also discovered increased proliferation, differentiation and
remodeling of bioengineered human nucleus pulposus cells
after injection with PRP [47]. Sawamura et al. discovered
increased expression of mRNA proteoglycan core protein,
type II collagen, and reduced apoptotic cells compared to
placebo, in IVD nucleus in a rabbit model with a PRP
impregnated hydrogel. On completion of the study, MRI
revealed preserved disc water content and increased disc
height in degenerative discs treated with PRP compared to
placebo [48]. Porcine intervertebral disc cells cultured in
alginate beads were treated with PRP and compared to
platelet poor plasma (PPP). There was a mild activating
effect on cellular proliferation, and a significant upregula-
tion of collagen and extracellular proteoglycan concentra-
tion when compared to PPP. Interestingly, these findings
were more dramatic in the annulus fibrosis cells than in
those of the nucleus pulposus [49]. Surprisingly, despite
being common in both athletes and sedentary individuals,
PRP use in the spine is still limited.

Cartilage

Osteoarthritis (OA) is common and frequently encountered


in a sports medicine setting. OA features include inflamma-
tion, recruitment of cells which release pro-inflammatory
factors, and proteinase activation which leads to degeneration
and death of chondrocytes [50]. At present, there are limited
3 PRP: Tips for Application in the Musculoskeletal 77

options for treating mild to moderate arthritis. Most tradi-


tional treatments are directed toward controlling the symp-
toms rather than correcting the biochemical environment of
the disease process. Disease progression is believed to result
from an imbalance between pro-inflammatory cytokines
(including interleukin [IL]-1a, IL-1, and tumor necrosis
factor-α) and anti-inflammatory cytokines (including IL-4,
IL-10, and IL-1ra) [50]. This cytokine imbalance is thought to
promote proteolytic enzymes that lead to cartilage destruc-
tion [51, 52].
There is some indication that the growth factors secreted
by the platelets in PRP have an effect on extracellular matrix
synthesis, chondrocyte metabolism and improved cartilage
healing in vitro [22, 53]. In vitro, PRP may have an anti-
inflammatory effect on chondrocytes, in part by its inhibitory
effect on IL-1β and NF-ĸB, a critical inflammatory regulator
in chondrocytes, and may also reduce chemotaxis [54, 55].
PRP may also up-regulate IL-1-receptor antagonist through
its up-regulation of mesenchymal stem cells (medicinal sig-
naling cells), which may then lead to retarding the progres-
sion of OA [50, 56]. Additionally, exposing synovial fibroblasts
of arthritic patients to PRP results in enhanced hyaluronic
acid production [57]. In human studies, intra-articular injec-
tion of PRP resulted in greater reduction in pain and
improvement in knee function when compared with hyal-
uronic acid (HA) injections in younger (less than 50 years)
patients with early OA at 6 month follow-up (P < .005), but
HA and PRP yielded similar results in more advanced OA
and older patients [58]. Multiple studies have demonstrated
statistically significant improvements in pain and function
following injection of PRP for knee and hip OA, with results
stable for 24 months in one group [59–62].
In summary, with increased PRP application in osteoar-
thritis, we are seeing more positive data both in vitro and
in vivo. However, this information has produced new ques-
tions. PRP results may fluctuate from patient to patient
depending on individual variability, including baseline plate-
let count, level of chondropenia, activity level, underlying
78 S. Sampson et al.

medical history, immune response and psychosocial factors.


Furthermore, OA is a heterogeneous degenerative disease,
and this also influences treatment outcomes. Important ques-
tions which must be addressed include whether and how PRP
balances the anabolism/ catabolism process, and what the
ultimate effects of PRP on synovium are Perhaps the ideal
PRP cocktail in OA should be more anti-inflammatory with
less pro-inflammatory cytokines, given the underlying physi-
ological processes of OA. Future research aims to isolate
cytokines contained in PRP termed “autologous conditioned
serum” to specifically inhibit IL-1 and TNF-α and down regu-
late MMP-13. Autologous Protein Solution Inhibits MMP-13
production by IL-1b and TNFα-Stimulated Human Articular
Chondrocytes [63].

Rehabilitation Following PRP Injection


There has been great variability, but little evidence, to guide
clinicians in rehabilitation after PRP injections. Most of the
rehabilitation protocols available relate to healing of tendons
and soft tissues. The protocol should follow the phases of tis-
sue healing (Fig. 3.6), as it provides insight to what is occur-
ring at a cellular level, and can help guide the rehabilitation
process.
Each phase is dependent on the preceding phase to work
successfully. Phase I, the inflammatory phase, generally lasts
48–72 h. In this phase, debris is removed from the damaged
tissue, as cytokines and growth factors are recruited to assist
in the healing process. Phase II, the proliferative phase, gen-
erally lasts 48 h to 6 weeks. During this phase, there is proteo-
lytic degradation of damaged tissue and chemoattraction of
neutrophils, lymphocytes, and macrophages to the area. There
is also introduction of fibroblasts, which form a new extracel-
lular matrix which leads to wound contraction. There is
increased neovascularization in the proliferative phase of
wound healing, which recedes as Phase III begins. Phase III is
the maturation phase, when functional tissue is laid down
3 PRP: Tips for Application in the Musculoskeletal 79

Proliferative phase
Inflammatory phase Maturation phase

• Collagen
accumulation
• Remodeling

Granulation Wound
Inflammation
tissue contraction

0.1 0.3 1 3 10 30 100

Fig. 3.6 The Phases of Tendon Healing (Adapted from Mautner


(2011) and Kumar (2004). Used with permission from Kumar et al.
[75])

[64]. Phase III begins around the sixth week. During this
phase, new extracellular matrix is laid down primarily through
accumulation of Type I collagen, the foundation of healthy
tendons [64]. This phase lasts for several months, even up to
a year. Thus, measuring outcomes from PRP injections should
require adequate time for healing to occur.
Based on the above framework, rehabilitation following
PRP injections can be divided into three phases as well
(Fig. 3.7). The first is the acute phase immediately after a PRP
injection is performed. The principal treatments of this phase
are pain control and tissue protection. It is recommended to
minimize excessive motion of the involved area and allowing
local platelet activation while avoiding disruption of the
fibrin plug during this period. This can range from limiting
weight bearing or resistance to frank immobilization. There
are no studies to demonstrate that immobilization enhances
80 S. Sampson et al.

Post PRP Rehabilitation


PHASE LENGTH RESTRICTIONS REHABILITATION
OF TIME
Phase I Days 0-3 • Consider NWB or • Relative rest
protected WB for • Activities as tolerated; avoiding
Tissue Lower extremity excess loading or stress to treated
protection procedures, area
especially if in pain • Gentle AROM
• No weight training
• Avoid NSAIDS
• Limited Ice
Phase II Days 4-14 • Progress to FWB • Light activities to provide motion
without protective to tendon; aerobic exercise which
Early Tissue device avoids loading of the treated
healing; • Avoid NSAIDS tendon
facilitation of • Gentle prolonged stretching
collagen • Begin treatment on kinetic chain/
deposition adjacent regions
-- Glutei strengthening
-- Core strengthening
Weeks 2-6 • Avoid eccentric • Progress weight bearing activities
exercises • Low weight, high repetition
• Avoid NSAIDS isometrics ( pain scale <3/10)
• Avoid Ice -- Open kinetic chain (OKC)
activities
• Soft tissue work to tendon with
CFM, IASTM,
• “Dynamic” stretching
Phase III Weeks 6-12 • Eccentric exercises ( keep pain
scale <3/10)
Collagen -- 2 sets of 15 repetitions
strengthening • Closed kinetic chain (CKC)
activities
• Plyometrics; proprioceptive
training and other sport-specific
exercises
• Progress weight bearing activities
and consider return to sport if
pain <3/10
Months 3+ Reassess improvement; if • Progress back to functional sport
not > 75 % improved specific activities with increasing
consider repeat injection and load on tendon as pain allows.
return to Phase I

Fig. 3.7 The 3 Phases of PRP Rehabilitation

outcomes; indeed, the negative effects of prolonged immobi-


lization are well known [65].
After the acute inflammatory phase subsides, the next sev-
eral weeks of rehabilitation should focus on preparing the
body for new tissue formation during the proliferative phase.
Controlled motion to the involved tendon is very important
in the first 2 weeks after PRP treatment. Virchenko and
Aspenberg demonstrated that, if botulinum toxin was admin-
istered to a muscle at the same time as PRP administration to
the tendon, there was no platelet effect noted at the 14-day
3 PRP: Tips for Application in the Musculoskeletal 81

mark, compared to the control group with no botulinum


toxin injection. This study demonstrates that mechanical
stimulation helps drive tendon healing [66].
During the early rehabilitation program, gentle, prolonged
stretching of the involved tendon is recommended. Preventing
future overload on the tendon is imperative to avoid re-
injury. Therefore, all patients who undergo lower extremity
procedures will work on the glutei, especially gluteus medius,
strengthening, as many lower extremity injuries have been
associated with weakness of this muscle [67]. In general, from
2 to 6 weeks, we recommend soft tissue work to the involved
tendon and beginning low intensity strengthening exercises,
such as low weight, high repetition isotonic exercises, to pro-
gressively increase the load placed on the tendon.
The maturation phase begins around week 6. This is the
time when eccentric exercises of the involved tendon are
introduced. Eccentric exercises can be effective in chronic
tendinopathy [68, 69]. It is possible that early eccentric activ-
ity may induce a cessation of the regenerative cascade by not
allowing proper angiogenesis to occur and by putting too
much load on the tendon too early. As healthy collagen
begins to accumulate, the mechanical effects of heavier load
exercises should improve the strength of the tendon. Most
human clinical trials on PRP allow progressive return to
activities as symptoms decline. However, the available litera-
ture does not specifically address when those activities were
resumed. Most of the research on PRP and the authors’ own
clinical experience has demonstrated improvement in pain
and function in a linear fashion over the first 6 months [34].
Hopefully, with more studies on PRP, we can continue to
refine the rehabilitation protocol following the procedures to
help optimize patient outcomes.

Tips and Concerns


Many physicians utilize image guidance, musculoskeletal
ultrasound, fluoroscopy or, less commonly, CT or MRI to
ensure accurate needle placement before injecting PRP
(Fig. 3.8). The authors believe image guidance is absolutely to
82 S. Sampson et al.

Fig. 3.8 Ultrasound guided Knee Aspiration of the Suprapatellar


Bursa

verify proper intra-articular placement. Empiric data sup-


ports the benefit of placing the PRP in the exact location of
pathology in tendon, ligament or muscle. The use of a large
gauge needle, both to obtain the pre-spin whole blood (17G-
18G) and to deliver the PRP product (22G), may prevent
early activation of the PRP. Many physicians have success-
fully used smaller gauge (25G) needles to deliver PRP prod-
ucts [18]. This presents an area deserving of further research.
Often, injecting a patient with a 25 g needle may not neces-
sarily result in less pain from the procedure, as increased
pressure may be experienced by the patient. Therefore, the
authors often utilize a 22G needle with varying length.
While the natural and autologous nature of PRP to self
repair is appealing, its premise is not without limitations. By
non-selectively concentrating a cocktail of both pro- and
anti-inflammatory cytokines with both anabolic and catabolic
activity, we are introducing a biologic agent that is not cus-
tomized. In general, patients have already failed one or more
treatment cycles, and are experiencing a state of failed heal-
ing response. The hope is that concentrating the platelets and
re-introducing them to the area of injury will stimulate repair.
However, there may be inherent biologic limitations that
3 PRP: Tips for Application in the Musculoskeletal 83

inhibit healing. For example, a patient’s activity level, base-


line platelet count, inflammatory state, underlying morbidi-
ties, age, hormone and nutritional status may hinder recovery.
Therefore, many adjuncts are emerging as possible comple-
ments to PRP to further individualize the therapy and pro-
mote healing.
Intra-articular human growth hormone (HGH) may have
a synergistic role with stem cells and growth factors. Through
the JAK-STAT signaling pathway, growth hormone stimu-
lates the production of insulin-like growth factor 1 (IGF-1)
[70]. IGF-1 down regulates pro-inflammatory cytokines such
as TNF-alpha and (IL)-1beta to accelerate the regenerative
process of injured skeletal muscle, balance the inflammatory
response, and limit fibrosis [71]. Dunn et al. illustrated
increased cartilage growth from intra-articular injections of
HGH [72]. Kim et al. showed that intra-articular injections of
HGH with HA in a rabbit model of collagenase induced
arthritis resulted in shorter duration and lower severity of
lameness, with less cartilage damage and superior histopatho-
logical scores [73]. However, long term trials are needed to
better understand its potential safety and efficacy and its
clinical use is not recommended by the authors. Additionally,
testosterone is depleted in many patients suffering with mus-
culoskeletal pain in both genders. Future applications may
correct underlying metabolic deficiencies that contribute to
unhealthy aging that may optimize results in biologic thera-
pies. However, there is both controversy and limited long
term follow up studies to document the safety and efficacy of
these therapies either alone or in combination with PRP.

Expert Opinion
Among the earliest users of PRP in musculoskeletal disease,
the authors have seen good outcomes in challenging condi-
tions. However, PRP is not a first line option for most
patients, and should generally be considered when traditional
measures fail. However, every day our clinics are filled with
patients who are told they have no conservative options
available and must “live with the pain.” These patients, which
84 S. Sampson et al.

we term “Pioneer Patients”, are driving change in our field.


They are seeking out cutting edge therapies and indepen-
dently researching alternative options in clinics around the
globe. With the baby boomer population and projected
expanded lifetimes, there exists a great void in treatment
options to encourage an active lifestyle. While most of these
patients are in fact not elite athletes, they represent a “Vision/
Action” personality type, and require physical activity as a
“medication” to maintain balance, sanity and well-being. PRP
is by no means a panacea, and carries many limitations. Most
likely, we will look back at PRP as the earliest generation
autologous bedside biologic that opened the gate for more
advanced remedies. Cellular medicine offers tremendous
promise not only for orthopaedics, but for many other disci-
plines, including cardiovascular disease. The non-selective
milieu of PRP through whole blood centrifugation is a rather
primitive concept that unquestionably will be refined. While
the replication of the body’s natural balance of growth fac-
tors used synergistically may work best; recent studies have
shown that isolating or blocking particular cytokines may be
optimal for particular conditions. For example, PRP pro-
motes axon growth in spinal cord tissues through increased
IGF-1 and VEGF, but TGF-b1 in PRP exerts negative effects
on axon growth [74]. In the near future, we expect differing
conditions to require specific cocktails of cytokines and vary-
ing PRP preparations.

In general, PRP offers promise for recalcitrant tendinopa-


thies, cartilage disorders, and degenerative spine conditions.
However, PRP seems to work best in mild to moderate pathol-
ogy in active individuals, with early intervention being optimal.
For patients with severe musculoskeletal disease, more pow-
erful biologic therapies such as Bone Marrow Concentrate
(BMC) are being applied. With its mesenchymal and hema-
topoetic stem cell populations and abundant growth factors,
BMC therapy exhibits immunomodulatory, anti-inflammatory,
chondrogenic and proliferative qualities. BMC likely repre-
sents the next generation of autologous cellular therapy. While
3 PRP: Tips for Application in the Musculoskeletal 85

stem cell based therapies offer great promise, its research and
practical applications are in their infancy.

Conclusions
Because of its autologous status, ease of use and preparation,
and given its safety profile, PRP is gaining popularity and is
introducing physicians and the community to the realm of
biological therapies. Most likely, PRP use will persist for years
to come. However, it may be recommended for particular
diagnoses or may serve as an adjunct for other autologous cell
based therapies such as Bone Marrow Concentrate (BMC),
Fat Grafts or Adipose Derived Stem Cells. Much research is
still needed to elucidate the optimal concentration, need for
activation, leukocyte and RBC presence, timing and fre-
quency of injections, specific clinical indications, post injec-
tion rehabilitation, treatment adjuncts, and the role of
nutrition, hormone optimization, medical history and psycho-
logical factors. The authors have had good outcome using
PRP in thousands of patients while continually exploring new
ways to improve outcomes. Growing worldwide collaboration
with controlled trials is necessary to advance our understand-
ing of musculoskeletal disease, and to institute novel biologi-
cal therapies to maximize healing. Although challenging to
conduct robust trials in a clinical setting, practitioners should
collaborate with colleagues, analyze and share data and pub-
lish work to further establish and advance biologic medicine.

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rabbit model of collagenase-induced osteoarthritis. J Korean
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74. Takeuchi M, Kamei N, Shinomiya R, et al. Human platelet-rich
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p. 87–118.
Chapter 4
PRP in Tendons and Other
Non-bone Tissues
Sebastiano Vasta, Rocco Papalia, Vincenzo Denaro,
and Nicola Maffulli

Introduction
Though highly debated, platelet rich plasma (PRP) has been
increasingly used in all the fields of orthopaedic and trauma
surgery. Its mechanism of action is still unclear, and no clear
evidences of its effectiveness are available. PRP is supposed
to act as a biological healing enhancer, and is considered to
contain platelets and, in some formulations, white blood cells.
Platelets do not have nucleus but they contain small granules
rich in growth factors (GF) responsible for hemostasis and

S. Vasta, MD • R. Papalia, MD, PhD • V. Denaro, MD


Department of Orthopaedic and Trauma Surgery,
Campus Bio-Medico University of Rome,
Via Alvaro del Portillo 200, Rome, Italy
N. Maffulli, MD ()
Department of Musculoskeletal Disorders, Faculty of Medicine,
Surgery and Dentistry, University of Salerno, Salerno, Italy
Centre for Sports and Exercise Medicine, Barts and London School
of Medicine and Dentistry, Queen Mary University of London,
London, UK
e-mail: n.maffulli@qmul.ac.uk

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 93


Practice, DOI 10.1007/978-1-4471-7271-0_4,
© Springer-Verlag London 2016
94 S. Vasta et al.

healing. Many factors are involved in triggering the degranu-


lation of GF. Thus, not only the amount of platelets matters
regarding the effectiveness of PRP, but also other substances
coming together with the plasma.
Bone, ligaments and cartilage respond differently to PRP.
Therefore, the current available evidences are controversial.

Tendons
The incidence of tendinopathies is rising up since the increas-
ing popularity of playing sports among the general popula-
tion and also among sedentary individuals. Chronic
tendinopathy is a major problem, since the healing process
often produces a painful tissue with abnormal histological
features. Conservative measures are the main treatment
option for tendinopathies. Eccentric exercises, Extracorporeal
Shock Wave Therapy, peritendinous injections produce vari-
able outcomes. Surgery is considered the last available option
when nonoperative management has failed, though even the
outcomes of surgery are unpredictable. Tendinopathies are
supposed to result from an unbalance between external
stimuli and adaptive changes. Traditional treatments are
often unpredictable; thus, great efforts have been made to
enhance the healing process. Studies on animal models
showed that PRP is able to lower the COX-1 and COX-2
expression, as well as PGE2, which is involved in pain produc-
tion, decreased cell proliferation, and collagen production
[40]. PRP is able to increase cell number, pro- mote progeni-
tor cells differentiation and increase collagen fiber density,
thus restoring the normal tendon tissue architecture [3, 10, 33,
35, 38]. Clinical use of PRP is widespread, though its effec-
tiveness is still controversial. A recent meta-analysis on con-
trolled studies pointed out that there are no evidences
supporting or hindering the clinical use of PRP. Pooling data
by sites of tendinopathy showed mid-term (36 months) ben-
eficial effects on pain, comparing L-PRP (leukocite rich
PRP) to other existing methods [1].
Chapter 4. PRP in Tendons and Other Non-bone Tissues 95

Achilles Tendinopathy

A recent systematic review [14] on PRP and Achilles tendi-


nopathy analyzed twelve studies, 11 case-series and only one
double blind randomized controlled trial (RCT). All the level
IV case series reported positive results for PRP. The RCT [13]
compared two groups of 27 patients, one treated with PRP
and the other with saline solution. After the injection,
patients followed a standardized rehabilitation program
based on eccentric exercises. The two groups showed no dif-
ferences at any follow-up (up to 1 year), both in clinical out-
come and in ultrasound evaluation.

Patellar Tendinopathy

Two RCTs and one prospective cohort study are available,


reporting the outcomes of PRP injection in patellar
tendinopathy.
Vetrano et al. [37] in 2013 reported the outcomes compar-
ing two groups of patients. One (PRP group = 23 patients)
underwent 2 autologous PRP injections under ultrasound
guidance over 2 weeks, and the other group (ESWT group =
23) received 3 sessions of focused extracorporeal shock wave
therapy. At 12 months, therapeutic injections of PRP resulted
in better clinical results compared with focused ESWT in
athletes with patellar tendinopathy.
Dragoo et al. [15] in a 2014 RCT reported on 23 patients
with patellar tendinopathy on examination and Magnetic
Resonance Imaging (MRI). Patients were randomized to
receive ultrasound-guided Dry Needling alone (DN group;
n = 13) or with injection of leukocyte-rich PRP (PRP group;
n = 10), along with standardized eccentric exercise.
The PRP group experienced significantly better outcomes
compared to the DN group at 12 weeks, but the difference
between groups was not significant at ≥26 weeks. The authors
concluded that even though the PRP can lead to better clini-
cal results in the short time, its benefits dissipate over time.
96 S. Vasta et al.

Filardo et al. [17] compared, though in a non-randomized


fashion, 31 patients with chronic jumper’s knee. Fifteen
patients underwent multiple PRP injections and physiother-
apy, after failure of other conservative or operative measures.
Sixteen patients were primarily treated only with physiother-
apy. Three PRP injections were performed 2 weeks apart into
the site of patellar tendinopathy. Though patients underwent
to PRP injections showed statistically significant improve-
ment in all scores when compared to the control group, no
significant differences were observed at 6 months after the
intervention.

Lateral Epicondylitis

Krogh et al. [24] in 2013 published a double blind RCT. Sixty


patients with evidence of chronic Lateral Epicondylitis (LE)
were randomized to three groups: injection of PRP, saline, or
glucocorticoid. After 3 months, patients were evaluated using
the Patient-Rated Tennis Elbow Evaluation (PRTEE) ques-
tionnaire. The results showed no superiority of PRP or gluco-
corticoid compared to saline in lowering pain level.
Glucocorticoid showed a short-term positive effect on pain
(at 1 month) and reduced color Doppler activity and tendon
thickness compared with PRP and saline at US evaluation.
Thanasas et al. [34] in a RCT compared 14 patients treated
with a single injection of PRP and a homogenous group
treated by a single injection of autologous whole blood. Visual
Analog Scale (VAS) for pain and the Liverpool elbow score
were collected up to 6 months after treatment. No statistically
significant differences were shown in Liverpool elbow score,
while VAS was statistically lower for PRP group only at 6
weeks.
Creaney et al. [12] reported in a similar RCT outcomes
comparing autologous whole blood injection to PRP.
Seventy patients received the autologous blood, while 80
the PRP injection. At 6 months the PRTEE did not shown
any statistically difference.
Chapter 4. PRP in Tendons and Other Non-bone Tissues 97

Rotator Cuff

Rotator cuff tendinopathy is common, responsible for lower-


ing function quality of life. Despite the great improvements in
surgical techniques, we are still far from optimal treatments,
able to restore full function and to avoid recurrences. Current
re-rupture rate ranges from 11.4 to 94 % [7, 19, 30]. New
strategies to improve rotator cuff healing have been investi-
gated, including novel suture techniques. Single row and
double row configurations have been compared, but they did
show any significant clinical difference, though double row is
biomechanically superior [36].
Since new theories have been advanced about a “low heal-
ing capability” and “healing failure” rather than re-tear [11],
PRP could be a promising strategy to improve rotator cuff
healing. A huge number of studies have been published on
that issue, though evidences still remain weak. A recent meta-
analysis [41] on PRP and rotator cuff repair, suggests as there
are no favorable outcomes from PRP use, both in the overall
clinical scores and re-tear rate. There is a decreased rate of
recurrences among patients treated with PRP for small-
and medium-sized rotator cuff tears, but not for large- and
massive-sized tears.
Current available studies are reviewed in Table 4.1 [4–6, 8,
20–22, 29, 31, 32, 39].

Plantar Fasciopathy

Plantar fasciopathy is frequent in both athletes and sedentary


individuals. About 10 % of subjects managed with conserva-
tively do not respond, and further more invasive treatment
are required.
PRP injections have gained increasing interest also for
plantar fasciopathy. Currently three RCTs are available.
A randomized controlled trial by Kim and Lee [23] allo-
cated 10 patients to PRP injection and 11 to dextrose prolo-
therapy. No significant differences were evident at 6 months.
98 S. Vasta et al.

Table 4.1 Clinical trials reporting on outcomes of rotator cuff


repair enhanced with PRP administration
Level of Clinical Imaging
Repair evidence differences differences
Castricini [8] 2011 DR 1 NO NO
Barber [5] 2011 SR 3 NO Lower
retear
Jo [21] 2011 SUTURE 2 NO NO
BRIDGE
Bergeson [6] 2012 SR/DR 3 NO NO
Rodeo [31] 2012 SR/DR 2 NO –
Gumina [20] 2012 SR 1 NO Lower
retear
Weber [39] 2013 SR 1 NO –
Antuna [4] 2013 – 1 NO –
Randelli 2011 SR 1 Reduced NO
[29] pain in the
first post
operative
month,
SER higher
in grade I
and II tears
Ruiz-Moneo 2013 SUTURE 1 NO NO
[32] BRIDGE
Jo [22] 2013 SUTURE 1 NO Lower
BRIDGE retear
SR single row, DR double row

Monto [26] in 2014 published a RCT on 40 patients with


plantar fasciopathy. They were prospectively randomized and
treated with either a single ultrasound guided injection of
PRP or 40 mg of DepoMedrol. PRP treated patients showed
higher values in the American Orthopedic Foot and Ankle
Chapter 4. PRP in Tendons and Other Non-bone Tissues 99

Society (AOFAS) hindfoot score at 24 months compared to


the corticosteroid group.
Omar et al. [27] investigated 30 patients with plantar fasci-
opathy randomized in two groups: one group receiving PRP
injections, and the other steroid injections. At 6 weeks, the
PRP group showed significantly better outcomes than the
corticosteroid group.
A recent systematic review [18] on PRP injections for
chronic plantar fasciopathy concluded that there are promis-
ing evidences, though the quality of the available studies is
low, with very short term follow-up.

Knee Osteoarthritis
Knee osteoarthritis is disabling. Current available conserva-
tive treatments include physical therapy, oral drugs and intra-
articular injections, with variable effectiveness. Recently,
intra-articular PRP injections have been widely used, though
good quality level I evidence is lacking.
Four level I RCTs are currently available.
Cerza et al. [9] reported on 120 patients randomized into
two groups: 60 patients received 4 intra-articular injections of
PRP, and 60 patients received 4 intra-articular injections of
Hyaluronic Acid (HA) (20 mg/2 mL). Treatment with PRP
showed a significantly better clinical outcome than did treat-
ment with HA, with lower WOMAC scores up to 24 week
followup.
Patel et al. [28] in a double-blind RCT on a total of 78
patients (156 knees) with bilateral knee osteoarthritis were
randomized into 3 groups. Group A (52 knees) received a
single injection of PRP, group B (50 knees) received 2 injec-
tions of PRP 3 weeks apart, and group C (46 knees) received
a single injection of normal saline. At 6 month, the two
groups treated with PRP injections showed significantly bet-
ter outcomes compared with the saline group. There were no
differences between the single versus multiple PRP injection
groups.
100 S. Vasta et al.

Filardo et al. [16] randomised 109 patients with knee


osteoarthritis in 2 groups: 55 were treated with HA and 54
with PRP, with 3 injections 1 week apart. At up to 12 month
follow up, significant differences were found: only patients
with by low grade articular degeneration (Kellgren-Lawrence
scores up to 2) showed a favorable trend in favour of PRP.
Li and colleagues [25] published a RCT on 30 patients
with knee osteoarthritis, randomly divided into PRP group
and HA group, with a 1:1 ratio. At 6 month follow-up, no dif-
ferences were shown between the two groups.

Conclusion
PRP preparations contain high concentrations of platelets
that, once activated, undergo degranulation to release growth
factors with healing properties. PRP also contains plasma and
other growth factors implicated in wound healing, in addition
to thrombin, which has inherent biological and adhesive
properties [2]. PRP has an anti-inflammatory properties. In
addition, the GF contained in PRP can stimulate the healing
processes by acting as a scaffold, and thus attracting sur-
rounding cells [38].
Though it has been receiving increasing emphasis and its
use has been increasingly expanding in clinical practice,strong
evidences supporting the use of PRP have not been produced
yet. Many variables play a primary role in PRP effectiveness.
Some are related to PRP itself, and others to the various clini-
cal settings. Among the first, there are different preparation
methods, thus influencing the amount of platelets, of white
blood cells, and the presence of molecules that have a role in
activating platelets (e.g. thrombin). Moreover, PRP can be
administered as an injectable fluid or as a implantable fibrin-
containing patch or gel.
With regard to clinical setting, there are a number of fac-
tors that vary from one investigation to another. Features of
the patients (age, level of activity), previous treatments, num-
ber of PRP injection, post-treatment rehabilitation are some
of the possible variables.
Chapter 4. PRP in Tendons and Other Non-bone Tissues 101

Future studies should focus on more homogeneous groups


of patients, standardising the protocols for the use of PRP
and for post-injection rehabilitation.

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Chapter 5
Platelet Rich Plasma
in Articular Cartilage Lesions
Elizaveta Kon, Giuseppe Filardo, Berardo Di Matteo,
Giulia Venieri, and Maurilio Marcacci

Physiopathology of Articular Cartilage


Articular cartilage is a mesenchymal tissue composed of
65–80 % water and 20 % dry components, including extracel-
lular matrix (type II collagen and proteoglycans) and only
less than 10 % chondrocytes. Cartilage is an avascular, aneu-
ral and alymphatic tissue which receives nutrients by diffu-
sion from synovial tissue. It covers the articular surfaces of
bones and helps to spread the load on subchondral bone,
allowing shock-absorption, reducing shear stresses and pro-
viding lubrication to the articular surfaces. These biomechan-
ical properties are supported by type II collagen and
proteoglycans, which build up a complex network capable of
remaining stable for decades. These structures give cartilage
its resistance to compressive stress and its high elasticity [1].

E. Kon () • G. Filardo


Nano-Biotechnology Laboratory, Rizzoli Orthopaedic Institute,
Via di Barbiano n. 1/10, Bologna 40136, Italy
e-mail: e.kon@biomec.ior.it
B. Di Matteo • G. Venieri • M. Marcacci
Biomechanics Laboratory, Rizzoli Orthopaedic Institute,
Via di Barbiano n. 1/10, Bologna 40136, Italy

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 107


Practice, DOI 10.1007/978-1-4471-7271-0_5,
© Springer-Verlag London 2016
108 E. Kon et al.

Articular cartilage defects can be distinguished into: (a)


focal lesions and (b) degenerative defects. The former are
characterized by well-delineated margins and are usually
caused by trauma or, occasionally, by osteochondritis disse-
cans or osteonecrosis. The latter are more extended, with less
delineated margins, and are caused primarily by osteoarthri-
tis (OA) [2].
Cartilage tissue does not regenerate by itself. The reason
behind its poor healing potential is still unsolved, and there
are mainly two possible reasons for this [1]. The first one is
the absence of vascularization, which does not allow the nor-
mal inflammatory response to take place, thus differing from
other tissues where healing starts with an inflammatory
phase followed by other sequential processes leading to tis-
sue regeneration. The second one is the lack of undifferenti-
ated cells: the only cellular component is represented by
relatively few chondrocytes. Furthermore, the regeneration
process is more difficult in old patients because the number
of chondrocytes declines with age. Damaged cartilage can
undergo only a reparative process, which usually results in a
fibrous tissue with inferior biomechanical properties when
compared to the original one.
Two types of articular surface lesion can be differentiated:
superficial defects, which are limited to the cartilage layer,
and osteochondral defects, which also involve the subchon-
dral bone. The specific type of defect influences the repara-
tive process and the treatment approach required.
When a lesion occurs in the articular cartilage, the remain-
ing cells are not able to organize an adequate reparative
process, and fail to fill the defect and organize the
neosynthetized matrix correctly. This condition might be
responsible for insufficient biomechanical properties and the
development of osteoarthritic changes over time. In light of
this, several approaches, both conservative and surgical, have
been used to improve cartilage healing potential [3–5].
Among these treatment options, the local administration of
Platelet-rich Plasma (PRP) is an increasingly common way to
provide a biological stimulus to regeneration.
5 Platelet Rich Plasma in Articular Cartilage Lesions 109

Definition of PRP and Sources of Variability


PRP is an autologous blood-derived product, principally
composed of a high concentration of platelets. However,
there are several PRP formulations, which do not allow a
univocal definition of PRP to be established. There are sev-
eral procedures available to obtain PRP. The two main ones
are based on laboratory centrifugation and density gradient
cell separation [6]. The preparation method is a key factor in
the platelet (PLT) count. This is a crucial variable: if we con-
sider that the biological rationale of this kind of therapy is the
supplementation of growth factors (GFs), the number of
platelets determines the total amount of GFs administered.
Very different platelet concentrations have been reported in
the literature, up to 8 times compared with basal levels [7]. A
commonly accepted platelet concentration in PRP should be
approximately 400 % of the peripheral blood PLT count [8],
but several products are currently being prepared with lower
concentrations. However, the dose of GFs is correlated with
a better clinical outcome [9]. Production methods are also
important in determining cell content: in fact, some PRP
preparations, in addition to plasma and platelets, also contain
leukocytes and residual blood cells. In particular, disagree-
ment has emerged about the role of leukocytes: on the one
hand, leukocytes might have an important antimicrobial
effect; on the other, they can release matrix metallo-protein-
ases and reactive oxygen species which might cause cartilage
damage [10].
Other aspects of PRP are controversial: freezing storage
procedures may reduce platelet ability to release GFs [11]; the
activation method widely ranges among authors, from no acti-
vation counting on the in vivo activating effect of endogenous
collagen, to the employment of chemical agents (calcium chlo-
ride and thrombin are the most commonly used), biomaterials
or physical agents [12]. Furthermore, therapeutic protocols
vary among different authors with regards to the amount of
PRP used for each injection, the number of injections and the
time lapse, thus impeding direct study comparison in this field.
110 E. Kon et al.

Finally, the disease phase treated by applying PRP is crucial,


since the treatment may produce different effects in early or
well established osteoarthritis (OA) [13]. It is important to
define exactly the treatment target [14] to determine clearly
which specific stage of cartilage pathology can benefit the
most from this treatment. Standardization has not yet been
proposed on these issues, and further investigations are
needed to clarify the high number of still open questions [15].

Biological Rationale
The biological rationale behind PRP treatment is the topical
administration of platelet-derived GFs, a group of polypep-
tides normally involved in joint homeostasis, healing and
regenerating of the damaged tissue, including cartilage [16].
Platelets are traditionally known for their hemostatic and
coagulation functions, but they also play a key role in the heal-
ing mechanism of damaged tissues, primarily thanks to their
GFs. Platelets contain three types of granules: lisosomal gran-
ules, dense granules, and alpha granules. Alpha granules are
the source of GFs, including platelet-derived growth factor
(PDGF), transforming growth factor (TGF-β), platelet-derived
epidermal growth factor (PDEGF), vascular endothelial
growth factor (VEGF), insulin-like growth factor 1 (IGF-1),
fibroblastic growth factor (FGF), and epidermal growth factor
(EGF); they also contain cytokines and chemokines, which are
involved in stimulating chemotaxis, cell proliferation and mat-
uration, modulating inflammatory molecules and attracting
leukocytes [10]. Dense granules store ADP, ATP, calcium ions,
histamine, serotonin and dopamine, which also play a complex
role in tissue modulation and regeneration [10, 17]. Finally,
lisosomial granules contain acid hydrolases, cathepsin D and E,
elastases and lisozyme [18], and many other proteins whose
physiological role is not well characterized. Furthermore, the
plasma itself contains important molecules involved in the
healing mechanism of connective tissues, also contributing to
the platelet stimulus in tissue regeneration.
5 Platelet Rich Plasma in Articular Cartilage Lesions 111

Literature on the Clinical Application of PRP


Platelet-derived GFs have been administered both surgically
and conservatively in the management of cartilage lesions.
The most widely studied anatomical district is the knee, but
some publications are also available for the treatment of hip
OA and talar osteochondral lesions.

Knee Application

Concerning the surgical application of PRP, in 2003 Sanchez


et al. [19] used it in a cartilage avulsion of the knee in a
12-year-old soccer player. After the in situ re-attachment of
the fragment, PRP was administrated locally, obtaining an
excellent clinical outcome: 18 weeks later, the patient resumed
sport activity with full functional recovery. Encouraging clini-
cal results have also been described after the surgical applica-
tion of PRP in combination with scaffolds and mesenchymal
stem cells [20–22] but, in these cases, it is really difficult to
assess the actual contribution of PRP to the clinical outcome.
With regards to PRP application as conservative manage-
ment, several studies are available, including some high qual-
ity ones.
In 2008, Sanchez et al. [23] first reported the injection of an
autologous preparation rich in GFs (PRGF) in a retrospec-
tive observational study on 60 patients. Patients were divided
in two similar groups, both treated by 3 weekly intra-articular
injections, 30 PRGF and 30 with Hyaluronic Acid (HA).
Clinical evaluation was carried out at base line and at 5 weeks
of follow-up, with special focus on “stiffness”, “pain”, and
“function”. The results were encouraging, with higher
improvements in the PRGF group.
In 2010, Sampson et al. treated 14 patients for knee OA
with 3 injections of PRP 1 month apart. Patients were evalu-
ated at 1 year using the Brittberg-Peterson Visual Analog
Pain, Activities and Expectation score, VAS for pain, and
KOOS score. Ultrasonography was used to assess pre- and
112 E. Kon et al.

post-treatment cartilage thickness. There was a statistical


improvement in pain, both at rest and during physical activity,
even though no significant increase in cartilage thickness was
observed after PRP injections [24]. In the same year, Wang-
Saegusa et al. [25] published a prospective study on a large
cohort of 261 patients treated for uni- or bilateral knee OA,
who received 3 injections of PRP, 2 weeks apart. The patients
underwent clinical evaluation at 6 months follow-up, using
the WOMAC score, VAS, Lequesne Index, and SF-36, with
significant improvement in all the scores.
In 2011, Kon et al. [26, 27] first published the results of
PRP treatment with up to 24 months of follow-up. The pro-
spective study included 115 knees treated with 3 injections of
5 mL PRP, 1 every 3 weeks; the patients had a clinical history
of knee pain or articular swelling that lasted more than 4
months, and radiographic or magnetic resonance imaging
(MRI) signs of OA. Evaluation was performed at base line
and at 2, 6, and 12 months of follow-up using the IKDC objec-
tive, IKDC subjective, and EQ-VAS scores. No major compli-
cations were seen, except for a case of marked post-injective
pain and swelling, which resolved spontaneously after 2
weeks. Significant improvements were found in all variables
during the 6 months of follow-up. Eighty percent of the
patients expressed satisfaction with the treatment received.
However, a tendency to worsen was reported at 12 months.
To confirm the time dependence of PRP treatment, a subse-
quent evaluation at 24 months’ of follow-up was performed,
which underlined a marked decrease in all scores. The authors
estimated the median duration of the PRP effect to be 9
months.
A significantly better response was observed in young
male patients, and, relating to the grade of degeneration, in
patients with chondropathy alone. Good results were also
found in patients who had received previous surgical treat-
ment [28, 29].
Some investigations also compared the clinical efficacy of
PRP and viscosupplementation. The multi-center study pub-
lished by Kon et al. [13] compared PRP with low-molecular-
5 Platelet Rich Plasma in Articular Cartilage Lesions 113

weight HA (LWHA) and high-molecular-weight HA


(HWHA). The patients were divided into three homogenous
groups and underwent 3 weekly injections of PRP, LWHA, or
HWHA. Subjective IKDC and EQ-VAS were used for evalu-
ation at 2 and 6 months’ of follow-up. Significant improve-
ments were found for the PRP group at 6 months’ follow-up
for both scores, with encouraging results in particular in
patients with simple chondropathy; conversely, in the early
and severe OA groups no significant difference in clinical
outcome was observed. Furthermore, better results were
obtained in patients under 50. The same authors reported
another comparative study [30] on 144 patients with knee
cartilage degeneration, exploring the clinical efficacy of high-
concentrate leukocyte-rich PRP compared to low-concentrate
leukocyte-free PRP. The clinical improvement was significant
in both cases, but the PRP-leukocyte group suffered from
more swelling and pain reaction immediately after the injec-
tions. Spakova et al. [31] also published a comparative study
on 120 patients and reported better results for PRP treatment
in comparison to viscosupplementation.
Halpern et al. recently published the results obtained on
22 patients with early OA who were treated by a single injec-
tion of PRP and evaluated up to 12 months’ follow-up. A
significant decrease in pain was documented and also an
increase in functional recovery. However, no change in MRI
structural appearance were found after PRP injection [32].
Four clinical randomized trials have recently been per-
formed. The first one published by Sanchez et al. [33] com-
pared the efficacy of single-spinning leukocyte-free PRP to
hyaluronic acid (HA) in 153 patients evaluated by the
WOMAC score at 6 months’ follow-up. The most significant
result of this study was the higher percentage of responders
in the PRP group than in the HA group. Furthermore, the
study confirmed that this biological treatment produces
poorer results in moderate/severe OA, without a significant
difference when compared to HA. A second randomized trial
was performed by Cerza et al. [34], who compared a low-
concentrate PRP – Autologous Conditioned Plasma
114 E. Kon et al.

(ACP) – with HA; 120 patients were divided into two groups,
and underwent 4 weekly injections. After 6 months’ follow-
up, the ACP group showed a significantly better performance
than that of the HA group, with clinical superiority even in
patients with Ahlback grade 3 knee OA. A double-blinded
randomized controlled trial comparing leukocyte-rich PRP
and HA has recently been published by Filardo et al. [35],
who reported the preliminary results of the first 109 patients
treated (55 PRP and 54 HA) and followed up for 12 months.
The evaluation was performed by IKDC objective and sub-
jective, Tegner score, KOOS score and VAS for general
health status. A significant increase in clinical scores was
observed in both treatment groups, with no statistical inter-
group difference reported. A tendency towards better results
was found only in the subgroup of patients with low grade
cartilage degeneration (Kellgren Lawrence up to 2).
Therefore, the authors concluded that PRP should be limited
to early OA, but it should not be considered as a first line
treatment in moderate/severe OA.
Patel et al. [36] studied 78 patients with bilateral OA
(Ahlback grade up to 2). Both knees of each patient were
included in the study and three treatment groups were identi-
fied: the first one received a single injection of leukocyte-free
PRP, the second group 2 PRP injections, whereas the last
group received one injection of normosaline. Follow-up
evaluation was performed at 6 months: a superior clinical
outcome was evident for PRP-treated knees, with no signifi-
cant difference between one or two PRP injections. Moreover,
even in this study, low-grade degenerated knees (Ahlback
grade 1) presented far better results than those of more
severely degenerated knees.

Hip Applications

In 2011, Battaglia et al. [37] treated 20 patients with hip OA


with PRP ultra-sound-guided injection (3 intra-articular
injections 2 weeks apart). The patients included in this trial
5 Platelet Rich Plasma in Articular Cartilage Lesions 115

had very different degrees of cartilage degeneration; their


Kellgren-Lawrence score ranged from I to III. After 1 year,
the clinical outcome was positive, but worsening occurred
after 3 months up to the final evaluation, which suggests the
time-dependent effect of PRP.
Sanchez et al. [38] investigated 40 patients with unilateral
hip OA who received 3 weekly ultrasound-guided injections
of PRP. Evaluation for up to 6 months, using the WOMAC,
Harris, and VAS scores for pain, showed a significant reduc-
tion in pain both at 6 weeks and 6 months; furthermore, the
WOMAC score also showed a functional recovery. However,
11 of 40 patients did not have any beneficial effect after injec-
tive treatment, and required a metal resurfacing procedure.

PRP in Talar Osteochondral Lesions

Giannini et al. [22, 39, 40] introduced an innovative


arthroscopic one-stage approach based on autologous bone
marrow-derived mesenchymal stem cells (BMDCs), PRP
and, alternately, porcine collagen powder or hyaluronic acid
(HA) membrane to create a scaffold. The first clinical trial
involved 48 patients with focal lesions and followed-up for 24
months, using the AOFAS score. A significant improvement
was found up to the final evaluation. Most patients returned
to sport within 11 months. Clinical outcome was correlated
negatively with bigger lesion size and previous surgery. Five
second-look arthroscopies were performed at 1 year, and in 2
patients biopsies were taken, showing, at histology and immu-
nohistology, the presence of new cartilage tissue with varying
degrees of hyaline-like tissue remodeling. The overall find-
ings suggested that this novel approach might produce results
comparable to those of autologous chondrocyte implantation
(ACI), while avoiding the double surgery time and inherent
stress for the patient.
Mei-Dan et al. [41] prospectively evaluated the efficacy of
HA compared to PRP used injected in 30 patients (15 per
group) not responsive to previous conservative treatments.
116 E. Kon et al.

The patients were allocated to receive 3 weekly intra-


articular injections of HA or PRP, and were followed for up
to 28 weeks. The investigators used AHFS, AOFAS, and VAS
scores for pain, stiffness and function. PRP was more effec-
tive in controlling pain and re-establishing function com-
pared to HA.

Discussion
The clinical application of PRP is supported by increasing
interest and evidence, but debate is still inconclusive because
of the lack of robust clinical trials [42]. A critical review of the
available literature allows to gain a better understanding of
the potential and feasibility of applying PRP in the manage-
ment of cartilage pathology. Concerning surgical application,
it is not possible to draw definite conclusions about the effi-
cacy of this treatment. In many surgical trials, PRP has been
administrated as an augmentation, together with mesenchy-
mal stem cells [22, 39, 40] or scaffold [20–22]. Therefore, it is
not possible to understand the role of PRP in determining the
clinical outcome. Moreover, the studies available are often
just case series treating disparate conditions in biomechani-
cally very different joints. Further studies are needed to define
guidelines for using PRP in such procedures and to determine
the persistence of the effect of PRP, since at present the lon-
gest reported follow-up is only 24 months. Moreover, PRP
augmentation of hydoxyapatite-collagen scaffold has a nega-
tive effect on its ability to repair cartilage [43].
With regards to the conservative use of PRP for hip and
talar osteochodral lesions, few studies have been published.
Two case series [37, 38] deal with management of hip OA, and
confirmed the safety of PRP and its usefulness in providing
temporary symptomatic relief. However, given the lack of
randomized trials, there is no evidence to support PRP as a
first line treatment in degenerative pathology of the hip joint.
Concerning talar osteochondral lesions, the only comparative
quasi-randomized trial published [38] showed statistically
5 Platelet Rich Plasma in Articular Cartilage Lesions 117

superior results for the PRP group. However, the low number
of patients treated and the short follow-up evaluation do not
allow, even in this case, the use of PRP to be fully endorsed.
Conversely, with regards to PRP application as conserva-
tive management of OA of the knee, several trials are now
available. Musch still needs to be clarified, but the consider-
able amount of studies published allows us to draw some
conclusions [44, 45]. Firstly, PRP is safe, as confirmed by all
trials, which reported only minor adverse events, such as post-
injective swelling and pain. Looking at the clinical outcome,
trials confirmed the benefit of treatment, despite a gradual
worsening occurring over time. Filardo et al. [27] were the first
to calculate the average duration of the PRP effect, which is
estimated at about 9 months. Four randomized clinical trials
were recently published, three of them comparing PRP versus
HA and the last one against saline solution: although better
results have been found in the PRP group at 6 months’ follow-
up [33, 34], Filardo et al. [35] reported no overall difference
between PRP and HA in terms of clinical outcome at 12
months’ follow-up. Nevertheless, a tendency towards better
results was found in young patients with a lower degree of
cartilage degeneration, whereas moderate or severe OA show
less favorable outcomes without difference compared to vis-
cosupplementation: the response to PRP may therefore be
linked to the stage of the condition. No definitive conclusions
can be drawn about the possibility of applying this approach
to a specific phase of cartilage degenerative pathology, espe-
cially if we consider that, until now, patients treated in the
aforementioned randomized trials were not homogeneous in
terms of disease stages, showing from chondropathy to severe
OA. What emerges can be considered as just a “suggestion”,
i.e. to avoid the indiscriminate use of PRP, the application of
which should be reserved to patients who can obtain the best
results from PRP (young patients with less articular degenera-
tion), and only as second line treatment in case of no response
to other traditional conservative managements for the other
patients, taking care to inform them not to expect miracles
from this biological treatment.
118 E. Kon et al.

Conclusions
Many questions are still open concerning PRP treatment of
cartilage lesions. At present, no conclusive indications are
available about this topic because only a few and heteroge-
neous high quality trials have been published. The only aspect
to emerge with enough scientific evidence is the safety of the
procedure, but clear superiority compared to other traditional
treatments has not been fully proven [44]. Further studies
should allow to dispel these doubts, but the indiscriminate use
of PRP should be avoided. PRP should not be a first line treat-
ment for cartilage pathology, and should be used in patients
who, according to the current scientific evidence, can obtain
the best results from this approach. The best PRP formulation,
the best protocols of administration, and the profile of the
patient and lesion that can benefit more from this biological
therapy are aspects which remain to be clarified.

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Garcia Gutierrez A, Albillos J, Gárate R, Aguirre JJ, Padilla S,
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the short-term treatment of symptomatic knee osteoarthritis.
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A, De Biasi G, Ciuffreda M. Comparison between hyaluronic
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2822–7.
35. Filardo G, Kon E, Di Martino A, Di Matteo B, Merli ML,
Cenacchi A, Fornasari PM, Marcacci M. Platelet-rich plasma vs
hyaluronic acid to treat knee degenerative pathology: study
design and preliminary results of a randomized controlled trial.
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with platelet-rich plasma is more effective than placebo for knee
osteoarthritis: a prospective, double-blind, randomized trial. Am
J Sports Med. 2013;41(2):356–64.
37. Battaglia M, Guaraldi F, Vannini F, Buscio T, Buda R, Galletti S,
Giannini S. Platelet-rich plasma (PRP) intra-articular ultrasound-
guided injections as a possible treatment for hip osteoarthritis: a
pilot study. Clin Exp Rheumatol. 2011;29(4):754.
122 E. Kon et al.

38. Sánchez M, Guadilla J, Fiz N, Andia I. Ultrasound-guided


platelet-rich plasma injections for the treatment of osteoarthritis
of the hip. Rheumatology (Oxford). 2012;51(1):144–50.
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bone marrow-derived cell transplantation in talar osteochondral
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Chapter 6
Platelet-Rich Plasma in Knee
Osteoarthritis in the Athlete
Mary Alexis Iaccarino and Joanne Borg-Stein

Introduction
Osteoarthritis (OA) is a degenerative disease of synovial
joints resulting from the combination of mechanical stress
and biochemical cellular changes which ultimately cause pain
and impair joint function. Osteoarthritis is the eighth leading
non-fatal burden of disease worldwide and a major cause of
disability [1]. The prevalence of symptomatic OA increases
with age: it is estimated that 18 % of women and 9.6 % of
men over age 60 have knee OA [1]. With increased longevity
in the population the burden and prevalence of OA is
expected to grow [2]. Furthermore, the epidemic of obesity
and resultant motivation to exercise, often through sports,
places more middle aged and older adults at risk of develop-
ing degenerative joint disease [3]. For the aging and former
athlete, OA may be a result of active adolescence and result
from prior ligamentous injury, meniscal trauma, or joint dis-
location. In a recent study, aging male athletes were 1.6 times
more likely to develop knee OA than controls [4].

M.A. Iaccarino, MD • J. Borg-Stein, MD ()


Department of Physical Medicine and Rehabilitation,
Harvard Medical School, Boston, MA, USA
e-mail: jborgstein@partners.org

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 123


Practice, DOI 10.1007/978-1-4471-7271-0_6,
© Springer-Verlag London 2016
124 M.A. Iaccarino and J. Borg-Stein

Goals in the treatment of knee OA are multiple and spe-


cific of a patient’s expectations. For the active adult, increased
mobility, sports participation, and pain reduction are the most
important end points. Younger athletes may want to defer
arthroplasty or other joint restoration procedures. Mechanical
joints have a limited lifespan and surgical cartilage restora-
tion requires multi-stage procedures and prolonged rehabili-
tation. In some individuals, comorbidities may preclude them
as surgical candidates. Non-surgical interventions, such as
corticosteroid injections, have a short duration, require
repeat administration, and have potential for local and sys-
temic side effects [5, 6]. Therefore, there is a need for a non-
invasive but effective treatment for knee OA. There is a
growing body of literature on the role of regenerative inject-
able therapies in the treatment of refractory knee pain [7–11].
Platelet-rich plasma (PRP) is a regenerative therapy that is
thought to promote healing by augmenting and accelerating
the natural healing cascade [12–15]. In this chapter we will
review the pathophysiology of knee OA, examine the current
evidence for PRP use in knee OA, describe clinical applica-
tions, and examine its future therapeutic role.

Pathophysiology of Knee Osteoarthritis


The human knee joint is composed of articular cartilage, sub-
chondral bone, a synovial membrane, synovial fluid, and a
joint capsule. Each articular component, along with the peri-
articular tendons, ligaments, and muscles aids in proper joint
function. Knee OA is an insidious and degenerative process
that can cause pain, instability, and ultimately joint failure.
Once thought of as a disease of cartilage and bone, any por-
tion of the knee including ligaments, tendons, and connectives
tissue may contribute. The exact pathophysiology of joint
degeneration is not completely understood and it is likely
multifactorial. However, the presence of abnormal mechani-
cal stress and changes in the normal biochemical articular
mileu are common contributing factors in this disease [16].
6 Platelet-Rich Plasma in Knee Osteoarthritis 125

Changes in the biomechanics of the knee joint are com-


monly observed in patients with osteoarthritis, and biome-
chanical stress is thought to be a main contributor to the
degenerative process [17]. In the healthy knee joint, load
bearing affects cartilage deposition and degradation. When
ambulating, the areas of greatest load in the normal knee are
the anterior medial and posterior lateral compartments and
there is a corresponding area of thick articular cartilage to
offset the load. Ligament or tendon injury, trauma, muscle
weakness, and weight gain can alter the normal gait pattern
changing the region of greatest load bearing. Healthy chon-
drocytes are able to adjust to load bearing. However, with
aging, injury, and trauma, cartilage becomes thin and begins
to degenerate. In the athlete, a commonly example of biome-
chanical stress occurs after an anterior cruciate ligament
(ACL) injury. There is a shift in the peak knee adduction
moment and increased internal tibiofemoral rotation, alter-
ing the load bearing areas of the knee during ambulation [18].
Imaging studies in patients with ACL injuries reveal thinning
of cartilage in the new areas of maximal load [19]. In multiple
studies of the development of knee OA after ACL injury,
peak knee adduction movement can be used to predict sever-
ity and progression of OA [20–22].
Joint inflammation also contributes to OA. It is the prod-
uct of a complex biochemical pathway that ultimately shifts
the joint milieu from that of anabolic activity with collagen
and extracellular matrix production to one of catabolic activ-
ity with expression of biomolecules that lead to degeneration.
Interlukin-1β is a potent pro-inflammatory cytokine. It is
thought to play a role in the breakdown of the chondrocyte
extracellular matrix contributing to the procatabolic effect
seen in OA [23]. Also implicated in OA are matrix metallo-
proteinases (MMPs), a group of matrix degrading enzymes
that act on collagen cleavage. Interlukin-1β, tumor necrosis
factor, and chemokines are all produced by chondrocytes and
activate MMPs. Inflammatory mediators such as prostaglan-
dins and nitrous oxide also activate MMPs, and decrease
biosynthesis of the normal extracellular matrix [24].
126 M.A. Iaccarino and J. Borg-Stein

Not limited to cartilage, inflammation is seen in the form


of synovitis. In histological samples of synovium from osteo-
arthritic joints, common downstream effects of inflammation
are evident including synovial hyperplasia, macrophage and
lymphocyte infiltration, fibrosis, and angiogenesis [25].
Several reviews of osteoarthritis pathogenesis implicate the
immune complement cascade and pathogen-activated Toll-
like receptors (TLRs) as contributors to OA and as potential
targets for disease modification [24, 25]. Many studies con-
tinue to explore the biochemical pathways involved in osteo-
arthritis to try and develop treatments that target molecular
disease progression. The role of PRP in targeting some pro-
inflammatory mediators is discussed below.
In the presence of biomechanical stress and activation of
catabolic pathways, the joint components begin to degrade
and undergo remodeling. Histologically, small fibrillation or
“nicks” form in the superficial cartilage. Through enzymatic
activity the nicks extend deep to the articular surface making
larger clefts. Eventually, this process extends to the subchon-
dral bone where remodeling is seen in the form of subchon-
dral cysts and periarticular osteophytes. Osteophytes are
outpouchings of bone that develop along the joint line, the
joint capsule, and the peripheral joint margin. Once thought
to be a compensatory mechanism to stabilize the joint, osteo-
phytes are now known to be associated with greater joint
misalignment. Along with osteophytes, cavitary lesions called
subchondral cysts form as a consequence of focal bone
resorption [26]. It is unclear if bone remodeling is a product
of cartilage degradation or an independent process that
occurs concurrently. However, bone remodeling is one of the
hallmarks of OA.

Nociception in OA
Cartilage is aneural. However, the remainder of the knee
joint is highly innervated. The joint capsule, tendons, retinac-
ula, fat pads, synovium, subchondral bone, and ligaments
6 Platelet-Rich Plasma in Knee Osteoarthritis 127

contain type III and type IVa fibers that contribute to pain
generation in OA. Through the release of neuropeptides sub-
stance P and calcitonin gene-related peptide (CGRP), these
nerve endings respond to noxious stimuli in and around the
joint. Cadaveric immunohistochemical studies show increased
presence of substance P and CGRP in degenerative com-
pared to healthy knees [27]. Substance P has also been identi-
fied in the lateral retinaculum, a common site of anterior
knee pain in young adults [28]. Mechanoreceptors may also
provide nociceptive feedback. In a study of arthroscopy in
two unanesthetized patients, injecting fluid into the free
patellar space to increase pressure was associated with sig-
nificant pain [29]. Similarly, the presence of effusion and
increased effusion size are associated with worsening knee
pain [30]. The periosteum is also innervated by sensory and
sympathetic fibers that express CGRP and tropomyosin
receptor kinase A, respectively. These fibers become progres-
sively less dense in deeper structures such as mineralized
bone and bone marrow [31]. However, an MRI comparison
study in painful versus non-painful osteoarthritic knee joints
found pain to be associated with larger, more edematous, and
poorly delineated bone marrow lesions suggesting bone mar-
row as another pain generator [32].

Clinical Applications of PRP for Joint


Osteoarthritis

Current Research and Evidence

The healing process is schematically divided in three stages:


inflammation, proliferation, and remodeling. Healing begins
with the release of inflammatory mediators, followed by
extracellular matrix rearrangement, and finally collagen pro-
duction and matrix maturation [33]. Platelet alpha granules
contain growth factors (platelet derived growth factor, insu-
lin like growth factor, vascular endothelial growth factor,
128 M.A. Iaccarino and J. Borg-Stein

and transforming growth factor β) that are involved in each


stage of the healing cascade. The injection of autologous
PRP into the joint space and surrounding painful soft tissues
delivers a concentrated dose of these growth factors, which
enhance the healing process and reduce pain [8]. An in vitro
study of osteoarthritic chondrocytes obtained from 6 patients
undergoing knee arthroplasty showed that PRP releasate
reduced inflammation [34] and inhibited gene expression of
pathways that are involved in OA pathogenesis, specifically
interlukin-1β [23]. In a study of synovial fibroblasts, PRP
enhanced production of hyaluronic acid, and stimulated
hepatocyte growth factor, which may play a role in suppress-
ing neovascularization of arthritic joints [35]. In osteoblasts,
exposure to concentrated platelets has been shown to
increase cell proliferation, supporting its role in bone regen-
eration [36].
Studies of in vivo application of PRP for knee OA report
varying results, with only a few randomized controlled trials.
Sampson et al reported a case series of 14 patients with pri-
mary or secondary knee osteoarthritis with each patient
receiving three PRP injections at 4-week intervals. At 1-year
follow-up, there was a significant reduction in pain at rest,
with moving, and with the knee bent. There were no adverse
outcomes and 8 of 13 patients felt they achieved their goal
with the injection [37]. Gobbi et al treated a group of 52
active patients with knee OA, and found a significant portion
had reduced pain after two injections of PRP and returned to
prior level of activity at 12 months follow-up [38]. This same
group found that at 2 years of follow-up and interval repeat
injections, PRP treated patients continued to have improved
pain control and mobility [39].
The use of PRP in conjunction with or instead of other
well-documented OA treatments is still being explored.
When compared to placebo (saline injection) PRP improved
symptoms at up to 6 months [40]. When compared to hyal-
uronic acid (HA), results have been favourable, with some
studies showing PRP to be as effective as HA and other
showing it to be more effective [41–43]. In a study by Filardo
6 Platelet-Rich Plasma in Knee Osteoarthritis 129

et al, 109 patients with knee chondropathy or OA received


either highly concentrated leukocyte-rich PRP or HA. After
3 weekly injections, the PRP and HA group had similar
improvement in Western Ontario and McMaster University
(WOMAC) Osteoarthritis Index at 3 and 6 months follow-up
compared to HA [41].
In 2012, Sanchez et al conducted the first multicenter,
double-blind, randomized controlled trial comparing PRP to
HA. The study of 176 patients with symptomatic knee OA
found that 38 % of patients treated with PRP had greater
than 50 % reduction in pain at 24 weeks compared to base-
line. This was significantly higher than the pain reduction
scores for patients treated with HA. Improvements in stiff-
ness and physical activity were also higher in the PRP group
but were not significant [44]. A study by Kon et al of 150
patients with cartilage degenerative lesions and knee OA
compared PRP to low molecular weight and high molecular
weight HA. The PRP and low molecular weight HA groups
had similar improvement in International Knee
Documentation Committee (IKDC) scores at 2 months. At 6
months, the PRP group had greater improvement on IKDC
than either HA group with the high molecular weight HA
group showing the least improvement [43].
These early clinical studies are encouraging and empha-
size the need for more, larger randomized controlled trials.
One difficulty in evaluating clinical trials is that the protocol
by which PRP is collected, prepared, and injected is not yet
standardized. Variations in technique may lead to results
inconsistency and the overall lack of a standardized protocol
for PRP preparation and administration makes evaluation of
one study to the next difficult. Despite this variability, most
studies of PRP in knee osteoarthritis show that PRP treat-
ment is well tolerated. Reported adverse events were limited
to post injection soreness, and this rarely deterred patients
from completing a course of treatment [37, 39, 44–46].
Therefore, as studies continue to define the efficacy of PRP,
physicians can be confident that it is a safe and well-tolerated
procedure.
130 M.A. Iaccarino and J. Borg-Stein

Patient Selection

Clinical trials and basic science knowledge have started to


determine patient selection parameters that may alter the
effect of PRP treatment. Age, severity of cartilage degenera-
tion or knee osteoarthritis, duration of symptoms, prior thera-
peutic interventions, and patient activity level may all be
relevant. When evaluating a patient for PRP, it is important to
consider these variable, and advise patients accordingly, so as
to set appropriate expectations for therapeutic benefit.
Patient age impacts PRP outcomes, with younger individu-
als having greater benefits. In a subgroup analysis of 150
patients with cartilage degeneration or OA, Kon et al found
that patients younger than age 50 had greater benefit from
PRP at 6 months follow-up than patients over age 50 and that
younger patients had a better response to PRP than HA. In
patients over age 50, HA and PRP had similar results [43].
Hypotheses for age related efficacy include a less robust
response of aging chondrocytes to growth factors and a lim-
ited propensity for healing and regeneration in elderly
patients [45].
Severity of osteoarthritis may also influence patient selec-
tion. Plain radiography, MRI, or arthroscopy may be used to
assess disease severity [47]. There are also many osteoarthri-
tis grading systems such as the Outerbridge criteria, IKDC
score, Kellgren-Lawrence classification, the International
Cartilage Repair Society (ICRS) scale, the Arthritis Impact
Measures Scale (AIMS), and the WOMAC that help charac-
terize disease severity. In clinic practice, plain radiographs are
a readily available and cost-effective means of assessing the
knee joint. In combination with the patient’s symptoms and
physical exam, radiographs are used to grade the severity of
knee OA. Kellgren-Lawrence classification is commonly used
as an inclusion criteria in studies of PRP in OA (Table 6.1).
Patients with chondral lesions and OA, Kellgren classifica-
tion I and II (Fig. 6.1), show a tendency for better outcomes
after PRP treatment. Patients with more severe disease
(Kellgren classification III) have some limited and
6 Platelet-Rich Plasma in Knee Osteoarthritis 131

Table 6.1 Kellgren-Lawrence classification of knee joint osteoar-


thritis [73]
Grade Criteria
0 Normal
I Minute osteophytes of doubtful significance
II Definitive osteophyte; joint space unimpaired
III Moderate diminution of joint space
IV Joint space greatly impaired; subchondral sclerosis
Adapted from Kellgren [48]

Fig. 6.1 Standing radiographs of the bilateral knees showing small


joint space narrowing on the right consistent with Kellgren-
Lawrence grade II

short-duration symptom relief but the effect is not as good as


in less severe joint degeneration [41, 43, 49]. Patients with
Kellgren type IV are excluded from most studies. The healing
and regenerative properties of PRP may be insufficient to
overcome the boney and soft tissue injury seen in severe joint
disease. However, the partial benefit seen in more advanced
osteoarthritis may be evidence of the short-term anti-
inflammatory and palliative effect of PRP [50]. Overall,
132 M.A. Iaccarino and J. Borg-Stein

patients with cartilage defects and mild to moderate OA


show greater improvement after PRP treatment than those
with severe OA and significant joint deformity [45, 47, 49].
Since PRP is an emerging therapy whose efficacy is still
not completely elucidated, other evidence based conservative
interventions for treating osteoarthritis should be used in
conjunction with or prior to PRP. Patients should be encour-
aged to pursue lifestyle modifications, including weight loss
and cardiovascular land-based or aquatic exercise programs.
Historically, exercise programs focus on closed chain and
isotonic exercises, quadriceps strengthening, knee flexibility,
and balance training [26]. The correlation between exercise
and improvements in knee pain and disability are well docu-
mented, but the causality is poorly understood [51–53]. In
exploring therapeutic regenerative therapies for osteoarthri-
tis, researcher have found that load bearing, as occurs in
exercise, can alter expression of MMPs and other degradative
enzymes in chondrocytes [54, 55]. In muscle and tendon,
mechanical loading may increase the expression of IGF-1 and
other molecules responsible for maintaining the extracellular
matrix [54]. The molecular changes that occur as a result of
load bearing may provide an explanation for the benefits of
exercise in OA.
Other modalities such as heat, cold and transcutaneous
electrical nerve stimulation (TENS) act as adjunctive thera-
pies for pain relief in knee OA [56]. Knee orthoses such as
unloading braces and lateral wedge insoles in genu varum
improve biomechanics and limit multidirectional instability
[26]. Pharmacotherapy for knee OA includes analgesics for
symptom relief. Acetaminophen, oral or topical non-steroidal
anti-inflammatories, and tramadol are common first line
medications. Alternative therapies include capsaicin, glucos-
amine, and chondroitin sulfate. Recently, COX-2 inhibitors
have fallen out of favor, but celecoxib is still prescribed in
some patients. Injectable therapies such as corticosteroids
have a short-term effect and may reduce inflammation.
Viscosupplementation, such as hyaluronic acid, is also shown
to be beneficial (as discussed above) [56].
6 Platelet-Rich Plasma in Knee Osteoarthritis 133

If patients continue to experience symptoms after conser-


vative measures, they should be evaluated for other poten-
tial pain generators and for possible PRP injection of the
affected knee. The examiner should elicit a thorough pain
history including onset, duration, severity, exacerbating and
alleviating factors, and referral sites. A meticulous physical
examination of the knee should be performed, including
evaluation of joint alignment, localization of pain, measure-
ment of range of motion, testing of joint laxity and instabil-
ity, and determination of strength. Evaluation in weight
bearing stance and gait evaluation should also be performed.
The hip joint should be assessed as a possible pain generator
or for concurrent arthritis. As stated above, knee radio-
graphs may help with diagnosis. However, there is a known
discordance between symptomatic knee osteoarthritis and
radiographic findings and, as such, the two are not mutually
exclusive [57].
Ultrasound imaging or MRI may be appropriate to exam-
ine soft tissues and identify other pain generators. Both
intra-articular and extra-articular structures should be con-
sidered [58]. In the knee, extra-articular sources of pain
include the proximal tibio-fibular joint, medial and lateral
collateral ligamentous attachments, pes anserinus attach-
ments, iliotibial band attachments, and insertions of the
patella and quadriceps tendons (Fig. 6.2). In some cases, a
diagnostic injection of local anesthetic may help determine
the exact pain source. Platelet rich plasma can be injected
into these extra-articular areas. However, if a large area
requires treatment, an alternative injectate such as prolo-
therapy dextrose solution may be preferred because a
greater volume can be used as opposed to PRP preparations,
which are in smaller quantities.
To a lesser extent patient selections parameters such as
gender and BMI should be considered, with females and high
BMI being associated with worse outcomes [45]. Currently,
there is no evidence to suggest that ethnicity, occupation, or
muscle weakness (factors that contribute to OA) play a role
in PRP efficacy. The impact of a prior surgical intervention
134 M.A. Iaccarino and J. Borg-Stein

ITB
b
PT ST
SM
PP SA
BF
SA
MM
LCL LM GR
MCL
LCL **
* MCL

ITB
GR
LR
MR
ST PT

Fig. 6.2 Representation of extra-articular pain generators of the


knee. (a) Anterior view. (b) Superior view. BF biceps femoris, GR
tendon of the gracilis muscle, ITB ileotibial tract, LCL lateral col-
lateral ligament, LM/* lateral meniscus, LR lateral retinaculum,
MCL medial collateral ligament, MM/** medial meniscus, MR
medial retinaculum, PP popliteus tendon, PT patellar tendon, SA
sartorius muscle, ST tendon of the semitendinosus muscle

on the treated knee is not well known. In a study of patients


with and without prior cartilage shaving procedure, the
non-surgical group performed worse on some but not all out-
come measures [38, 39]. In other studies, patients having
undergone extensive surgery of the knee joint were excluded.
Further studies are required to understand the impact of
prior surgical interventions.

Methods
Methods for administration of PRP are variable and, to date,
there is no consensus on the frequency of injection, method of
PRP preparation, and injection technique. Thus, the following
methodology is based on clinical studies of PRP application.
6 Platelet-Rich Plasma in Knee Osteoarthritis 135

Patient Preparation Patients should undergo a meticulous


physical exam to confirm knee OA and assess tender
periarticular pain generators that can be included in treatment.
Risks and benefits are thoroughly reviewed with the patient,
and written consent is obtained. Similar to other intra-articular
injections, risks include bleeding, infection, peripheral nerve
injury, allergy to sterilization products and local anesthetic,
and temporary stiffness. Patients who have had prior steroid
or viscosupplement injections should be educated that PRP
has been associated with greater post-injection soreness that
can last 2–7 days. Goals and benefits of the treatment include
reduced pain, improved function, and better joint stability.
Contraindications for PRP administration include active
infection and inability to comply with post procedure guide-
lines. Some studies have excluded patients with hematologic
disease, thrombocytopenia, anemia, coagulopathy or active
immunosuppressed state [41, 42, 44, 46]. However, these con-
traindications are relative and physician dependent. The
presence of a joint effusion is not a contraindication and
there is evidence that PRP may have a positive effect on
synovitis [34, 59]. However, it may be beneficial to aspirate
large effusions prior to treatment because the high volume of
fluid is a pain contributor, and may impede injection of PRP
into the intra-articular space.
In the setting of anticoagulation, an injection can be con-
sider if the International Normalization Ratio is less than 2.5
and the patient is advised of the potential increased bleeding
risk. In this situation, a needle no larger than 25-gauge should
be used. In the case of prosthetic knee joints, intra-articular
injections are avoided. However, peri-articular injections can
be considered as long as the patient and clinician recognize the
potentially devastating complication of hardware infection.

Injection Technique The patient is placed supine with the knee


in slight flexion. Under ultrasound guidance and using a
lateral approach PRP is injected into the suprapatella bursa
which lies between the prefemoral and prepatella fat pads.
136 M.A. Iaccarino and J. Borg-Stein

This is best visualized under ultrasound if there is a small


amount of fluid. If no fluid is seen, an alternative approach
may be at the superior medial corner of the knee piercing the
retinaculum and then entering the joint. Four to five milliliters
of PRP are injected intra-articularly. One milliliter of PRP
can be injected into any painful entheses that have previously
been identified on ultrasound. Although there is no evidence
to mandate the use of musculoskeletal ultrasound, it offers
significant procedural benefit. It can be used to help identify
extra-articular areas of enthesopathy and correlate them with
symptoms, it provides needle guidance, and it confirms the
delivery of the injectate to the desired locations. A sterile
technique is used to minimize risk of infection. The use of
local anesthetic prior to injection is not recommended. In
vitro studies show that local anesthetic alters tissue pH, which
may reduce the effect of PRP [13, 60]. Alternatively, a topical
vapocoolant spray can be used to reduce the pain of needle
insertion.

Post Procedure Care After an injection, patients are advised to


avoid hot tubs, Jacuzzis, and pools for the first 24–48 h to
reduce infection risk. Analgesics such as acetaminophen can
be used for post-procedure soreness. In some cases, tramadol
or opiates are needed. Non-steroidal anti-inflammatories
may impair the inflammatory phase of healing and therefore
should be avoided. Immediately post-injection, progressive
weight bearing as tolerated is recommended. If the procedure
is limited to an intra-articular injection then patients may
progress back to normal activity in a graded fashion as pain
permits, progressing to greater activity if they are pain free. If
a peri-articular injection (patella tendon or ligament enthesis)
is performed, then a more conservative activity progression is
recommended to allow for tissue healing. In the first week,
patients may perform light aerobic activity as tolerated
(walking or light cycling) and strengthening away from the
involved joint (core and upper extremities). Over the second
6 Platelet-Rich Plasma in Knee Osteoarthritis 137

and third weeks, they can incorporate light strength training,


but should avoid eccentric exercise. In weeks four through six,
they can restart eccentrics and progress to sports specific
training. Patients are advised not to progress to a more
strenuous activity if there is pain or synovitis with a less
strenuous activity.

Frequency There are no definitive guidelines on number or


frequency of injections. Several clinical trials have found up
to 12 months of improvement in knee pain with one injection
[46, 49]. Other studies have utilized a series of injections over
several weeks to months. In a randomized clinical trial,
Sanchez et al administered PRP injections weekly for three
consecutive weeks with greater than 50 % reduction in knee
pain for 6 months [44]. Other trials have followed a similar
protocol, and found symptom improvement at up to 12
months [41, 42]. Patel et al conducted a subgroup analysis of
outcomes of one injection versus two injections 3 weeks apart
and found no difference between the groups at 6 months [40].
Monthly injections have also shown significant pain relief at 6
and 12 months of follow-up [37, 38]. Gobbi followed 80
patients for 24 months. All patients received two injections 4
weeks apart. Two subgroups had repeat injections, one group
at 6 and 12 months, and the other group at 12 and 18 months.
Indications for repeat injections were to preserve outcomes
and not for symptoms recurrence. Both groups had continued
pain relief at 24 months [39].

The role of maintenance injections as compared to those


for reoccurrence of symptoms has not been well studied. The
literature outlined above shows favorable outcomes in both
cases. No adverse events have been reported as a result of
repeat injection. However, for some patients, the activity limi-
tations after the procedure, the time commitment for injec-
tion series, and the cost of multiple treatments may deter
them from a multiple injection treatment plan, particularly if
138 M.A. Iaccarino and J. Borg-Stein

the outcome may not differ. If patients fail to respond to a


series of injections, there is currently no evidence to support
the use of a second series of injections. In those cases, patients
should consider alternative treatment plans.

What Have We Learned?


There is currently good in vitro evidence to suggest that tis-
sue exposed to PRP has a propensity to undergo healing and
self-repair. Clinical trials are advancing to determine the role
of PRP in clinical practice. Short-term follow-up studies show
that PRP to be effective in reducing pain and increasing
activity in patients with knee OA. Long-term follow-up stud-
ies and randomized clinical trials are now underway to better
characterize how to incorporate PRP into patient care. To
date, there is variability in long-term outcomes in part result-
ing from variability in patient selection, severity of disease,
and methods of injection.
It is the responsibility of the clinician to select the appro-
priate patient for this treatment. Ideal patients have mild to
moderate symptomatic osteoarthritis. They lack significant
knee deformity and have not had complete failure of the
knee joint. Imaging studies can be helpful, in particularly,
weight bearing plain radiographs give a more accurate assess-
ment of the degree of OA. In general, younger, active patients
with a reasonable expectation of returning to prior level of
physical activity will have a better result. Every effort should
be made to ensure that symptomatic knee osteoarthritis is the
pain generator. If in doubt, perform a diagnostic injection to
clarify the diagnosis. Superimposed pain generators should
be ruled out, and the examiner should understand their rele-
vance in the overall clinical picture.
When PRP is the appropriate treatment, it should be
used as part of a comprehensive treatment plan [16, 61].
Patients should be encouraged to modify life-style factors
that contribute to knee osteoarthritis [62]. They should
6 Platelet-Rich Plasma in Knee Osteoarthritis 139

optimize body mass index, strength, biomechanics, sleep,


and nutrition [63, 64]. Behavioral or psychosocial factors
that cause or exacerbate pain should also be addressed [62,
65]. Clinicians need to have an honest discussion with
patients regarding the current state of knowledge of PRP
and its potential benefits. They should identify patients’
functional and athletic goals and discuss recovery time and
return to play to set realistic expectations for treatment. If
there is more advanced OA and the patient is not a surgical
candidate, consider viscosupplementation or possibly stem
cells. Possibly, athleticall active young adults may be better
served with stem cell augmentation but data on this is
lacking.
The method of PRP preparation may also contribute to its
efficacy.
Attention should be paid to the PRP preparation, includ-
ing the presence of leukocytes, the platelet concentration, and
means of platelet activation. Studies have used both leuko-
cytes poor PRP (pure-PRP) and leukocyte rich PRP in knee
OA. In theory, leukocyte poor preparations may be favorable
as in vitro studies of leukocytes in soft tissue injury show
negative outcomes in wound healing and repair [66, 67]. The
concentration of platelets is also important, and may vary
depending on centrifugation process [68]. An acceptable
platelet concentration is considered at least 2–3 times base-
line whole blood, ideally 6–7 times, while concentrations
greater than 10 times baseline are considered to be excessive
and potentially detrimental [69, 70]. The presence of endog-
enous type I collagen, agitation of platelets during centrifuga-
tion, and needle induced bleeding all act as endogenous
platelet activators. However, several large studies of the knee
have added exogenous calcium chloride to facilitate platelet
activation [40, 44, 46, 71]. While studies continue to explore
the most effective preparation method, practitioners should
be mindful that formulations vary and should critically
review their own preparations methods and those of pub-
lished research trials [69, 70].
140 M.A. Iaccarino and J. Borg-Stein

Conclusions and Future Implications

Platelet rich plasma and other regenerative injections repre-


sent a major paradigm shift and advancement in the treat-
ment of knee osteoarthritis when compared to intra-articular
corticosteroid injections. However, there remains a need for
strong, sufficiently powered, randomized controlled trials to
validate its use over other traditional injection treatments
(corticosteroid and viscosupplementation). Future research
will need to optimize growth factor enhancement, consider
the role of PRP alone and in combination with stem cells [72]
and scaffolds, as well as, determine optimal delivery method.
Our ability to reduce pain, improve function, and perhaps
enhance hyaline chondrogenesis over the long term needs to
be further studied.

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Chapter 7
Platelet Rich Plasma in Foot
and Ankle Surgery
Catie Cunningham, Amit Sood, and Sheldon Lin

Introduction

Platelet-rich plasma (PRP) typically contains a fivefold


increase in platelet concentration, and can be obtained from
autologous blood and/or bone marrow [1]. Alpha-granules, a
type of secretory organelle found in platelets, act as a reser-
voir for a number of different growth factors. In the presence
of trauma to bone, platelet activation leads to degranulation
of these alpha-granules, which ultimately results in release of
these growth factors localized to the site of injury (Table 7.1).
Platelet-derived growth factor (PDGF), insulin-like growth
factor (IGF) and transforming growth factor (TGF-β1) are
among some of the factors released, and play critical roles in
bone heeling [1, 2]. Human and animal studies have
demonstrated increased PDGF (four to sixfold), IGF-I (one
to twofold), TGF-β1 (three to fivefold), vascular endothelial
growth factor (VEGF) (four to sixfold), and epidermal
growth factor (EGF) (three to fivefold) levels in activated
PRP compared to peripheral blood [3, 4].

C. Cunningham, MD • A. Sood, MD • S. Lin, MD ()


Rutgers Medical school, 185 South Orange Avenue,
Newark, 07103 NJ, USA
e-mail: linss@njms.rutgers.edu

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 147


Practice, DOI 10.1007/978-1-4471-7271-0_7,
© Springer-Verlag London 2016
148 C. Cunningham et al.

Table 7.1 Effects of growth factors found in PRP


PDGF Angiogenesis, macrophage activation, chemotaxis and
proliferation of fibroblasts, collagen synthesis
TGF-β Proliferation of fibroblasts, collagen synthesis, bone
matrix formation, inhibition of bone resorption
IGF Chemotaxis of fibroblasts, proliferation and
differentiation of osteoblasts, bone matrix formation,
growth and repair of skeletal muscle
VEGF Angiogenesis, migration and mitosis of endothelial
cells, chemotaxis of macrophages and granulocytes,
vasodilation
EGF Proliferation and differentiation of epithelial cells
Adapted with permission from Mehta et al.
PDGF platelet-derived growth factor, TGF-β transforming growth
factor beta, IGF insulin-like growth factor, VEGF vascular endothe-
lial growth factor, EGF epithelial growth factor

PDGF stimulates collagen deposition and production of


extracellular matrix, enhances chemotaxis and proliferation of
fibroblasts, and initiates differentiation of osteoprogenitor cells
towards an osteoblastic lineage [5–8]. IGF acts in a similar man-
ner by enhancing proliferation and differentiation of osteo-
blasts, stimulating collagen syntheses, aiding in bone matrix
formation, and enhancing skeletal muscle repair [9–12]. The
concentration of TGF-β1 is almost 100 fold greater in bone and
platelets than in any other tissue, with osteoblasts containing
the greatest number of TGF-β1 receptors [13, 14]. TGF-β1
increases cell proliferation, collagen synthesis, and stimulates
cartilage matrix production while inhibiting bone resorption [1].
A dose-dependent relationship between the concentration
of these growth factors and the number of platelets has been
demonstrated [15, 16]. Thus, local levels of these critical fac-
tors can be greatly increased by delivering a highly concen-
trated volume of platelets at the site of pathology. The use of
PRP as a biological adjunct for bone and soft tissue aims to
optimize the heeling response in patients suffering from mus-
culoskeletal injuries.
Chapter 7. Platelet Rich Plasma 149

Foot and Ankle Sport Injuries: Epidemiology


and Biomechanics

Injuries to the lower extremities account for the majority of


sports-related injuries with 55–90 % occurring from the hip
to the toe [17, 18]. Ankle sprains may be the most common
injury in sports despite the many which go unreported [19–
23]. The incidence of injury in running and jumping sports
range from 10–15 % for the ankle and 3–15 % for the foot
[20, 24–27]. DeHaven et al. examined injuries over a 7-year
period including patients participating in sports from the
recreational to the professional level. Of 3,431 patients iden-
tified, 12 % had injuries involving the ankle while another
3 % involved the foot [24]. Zaricznyj et al. examined the
causes of sports-related injuries in 25,512 school children:
11.4 % had injuries to the ankle and another 6 % with
injuries to the foot [27].
Kannus et al. reported that soccer, running, and orienteer-
ing caused the greatest number of foot and ankle injuries in
patients presenting to their European sports medicine clinic
[25]. In a study looking at ankle injuries among United
States high school athletes, basketball and football had the
highest incidence of injuries [28]. At a national level, ankle
injuries account for nearly 16–29 % of all soccer specific
injuries [29, 30].
Injuries to the foot and ankle plague recreational and pro-
fessional athletes worldwide. Knowledge of the biomechanics
of the foot and ankle is important to understand why athletes
are prone to such injuries, especially those combined with
cutting or sliding maneuvers.
The foot and ankle consist of a number of joints working
together to provide stability through bony, ligamentous and
tendinous interplay. The axis of rotation of the ankle joint
may be estimated by following an imaginary line from the
medial malleolus to the lateral malleolus in a medial and
posterior direction. This axis is in roughly 82° degrees (range
74–94°) from the midline of the tibia in the coronal plane
150 C. Cunningham et al.

(8° varus). The axis of the tibial plafond is angled in about 3°


of valgus (range 2–10°). The angle between these two is the
talocrural angle: it measures 83° ± 4° and helps determine fibu-
lar length in fractures. Despite a narrower width of the mor-
tise and talus posteriorly, the talus remains congruent with the
mortise throughout full range of motion, resulting in an
obligatory external rotation of the foot, with dorsiflexion and
internal rotation of the foot with plantarflexion at the ankle.
Medial ankle stability is provided by the deltoid ligament
which resists valgus forces on the hindfoot and provides a
restraint to external rotation of the talus within the mortis.
Lateral ankle stability is provided by the lateral collateral
ligament complex. The anterior talofibular ligament provides
stability with plantar flexion of the ankle and resists inversion
of the talus as well as anterior translation of the talus with the
ankle in neutral alignment. The calcaneofibular ligament
comes into functional alignment with ankle dorsiflexion, and
functions to resist inversion of the talus as well.
The syndesmotic complex consists of the anterior inferior
tibiofibular ligament, posterior inferior tibiofibular ligament,
and the transverse ligament. It binds the distal tibia and fib-
ula together, and, with the stability provided by the deltoid
ligament, helps to maintain the talus within the mortise pro-
vided that the fibula is in anatomic length and rotation with
intact medial ankle structures. Syndesmotic disruption may
be recognized through an abnormal talar position within the
mortise. Medial clear space widening may also be visualized
on plain radiographs with or without stress testing. In the
presence of a deltoid ligament tear, talar instability is present
when at least 3–4.5 cm of the syndesmosis is disrupted above
the mortise [31]. An intact deltoid ligament, however, pro-
vides talar stability obviating the need for syndemotic fixa-
tion. The level at which the fibula is fractured also may not
reliably indicate whether syndesmotic stability is present,
since the interosseous tear may extend proximal to the level
of fracture [32].
The hindfoot consists of the talus and calcaneus which are
stabilized by the interosseous ligament. The articulation
Chapter 7. Platelet Rich Plasma 151

between these two bones is referred to as the subtalar joint


which permits inversion and eversion. The range is usually
between 30° of inversion and 15° of eversion, with marked
variability among individuals.
The midfoot consists of the navicular, cuboid, and three
cuneiform bones (medial, middle, and lateral). The transverse
tarsal joint consists of the talonavicular and calcaneocuboid
joints which permit abduction and adduction. With the hind-
foot in an everted position, the transverse tarsal joints are
aligned parallel to each other allowing greater motion at this
joint. This occurs at heel strike and creates a flexible foot
which absorbs energy. When the hindfoot is inverted, such as
during toe-off in the gait cycle, the transverse tarsal joints
become nonparallel, thereby stabilizing the joint and produc-
ing a more rigid foot for forward motion.
The forefoot consists of the five metatarsals and toe pha-
langes. The plantar aponeurosis originates from the calcaneal
tuberosity and inserts onto the base of the proximal phalan-
ges. Upon heel rise, the metatarsophalangeal joints dorsiflex.
This produces depression of the metatarsal heads and a pull
on the calcaneus, resulting in an elevated longitudinal arch
and inversion of the hindfoot, providing a stable foot at
toe-off.
The ankle joint, subtalar joint, transverse tarsal joint, fore-
foot, and plantar aponeurosis of the foot are linked together,
and play an important role in the function of the lower
extremity. If the linkage is disturbed, it may increase stress at
neighboring joints leading to loss of motion, pain, and degen-
erative changes [33].
Injuries to the tendons of the foot and ankle occur primar-
ily through repetitive loading and higher energy trauma.
Acute tendon ruptures may also be attributed to underlying
degeneration caused by overuse. When stresses to tissues
exceed those within physiologic range, the collagen bundles
of the musculotendinous unit begin to fail, ultimately result-
ing in gross failure of the tendon or displacement of the joint.
The extent of the injury is dictated by the amount of force
transmitted through the tendon, a function of the ratio of
152 C. Cunningham et al.

cross sectional area of tendon compared to that of muscle


[34]. This mechanism is especially pertinent when considering
injuries to the Achilles tendon, whose cross sectional area is
significantly smaller than that of the powerful gastrocnemius
muscle.

Foot and Ankle: Soft Tissues and Cartilage


Reconstructive surgery of the foot and ankle remains a chal-
lenge. Barriers to healing include a pattern of arterial net-
works with a reliance on small vessels to provide vascular
inflow, thin soft tissue coverage, a high level of back pressure
within the venous system, and high mechanical stresses acting
across the foot and ankle joints. These obstacles are magni-
fied in compromised patients (i.e. diabetes, vascular insuffi-
ciency), and pose a challenge to the treating physician.
Initiation of tendon and ligament healing is dependent on
blood clot formation and penetration of inflammatory cyto-
kines from surrounding tissue. In normal healing, as inflam-
matory cells are recruited to the site of pathology and
damaged tissue is removed, fibroblasts begin to proliferate
and new extracellular matrix is produced with type III colla-
gen and proteoglycans. Over time the amount of inflamma-
tory cells, proteoglycans, and type III collagen begins to
decrease, marking the beginning of the remodeling phase
which is important for restoring proper function to the dam-
aged tissue [35]. Tensile strength is increased, as cross-linked
type I collagen replaces type III collagen and as the collagen
fibrils change their orientation parallel to the axis of mechan-
ical stress.
Articular cartilage has little capacity for repair and regen-
eration. Hyaline cartilage defects repair by forming scar tis-
sue in the form of fibrocartilage from the subchondral bone.
This scar tissue is deficient in type II collagen, and has abnor-
mal proteoglycans with inferior biomechanical characteristics
and lower load bearing capacity [35]. This often results in
initial short-term recovery with later surface deterioration,
Chapter 7. Platelet Rich Plasma 153

commonly progressing to chronic pain, poor function, loss of


motion, and early onset osteoarthritis for the patient [34].
For these reasons, sports-related injuries of the foot and
ankle whether they relate to bones, tendons, or ligaments,
may develop impaired healing responses. The use of a bio-
logical adjunct such as PRP could potentially aid in the initia-
tion and propagation of the healing cascade by providing the
growth factors required for regeneration.

PRP: Any Evidence of Effectiveness?

Basic Science
Several in vitro studies have investigated the effect of PRP on
bone healing (Table 7.2). Oprea et al. sought to determine the
effect of PRP on recruitment and migration of rat bone mar-
row derived cells within a fibrin matrix [36]. A fibrin gel over-
lay was added to cultured cells, and PRP applied to its surface.
PRP application increased the leading front of migration by
25 %, the number of migrating cells threefold, and cell prolif-
eration twofold. Additionally, these cells exerted a significant
contractile force on the fibrin matrix which was directly
related to the migratory activity of the cells. Kanno et al. stud-
ied the effect of PRP on osteoblast differentiation in human
osteosarcoma lines: it had both a dose-dependent and time-
dependent positive effects on the expression of alkaline phos-
phatase, procollagen type I, and osteopontin genes [37].
Kruger et al. studied the effect of PRP on human subcorti-
cal progenitor cells [38]. These cells are involved in cartilage
healing using the microfracture technique. They found
increased cell migration and cartilage matrix formation at the
site of microfracture. In addition, gene expression analysis of
typical chondrocyte, osteogenic and adipogenic markers
showed that PRP induced chondrogenic differentiation with-
out osteogenic or adipogenic differentiation, suggesting a
possible role in cartilaginous healing.
154 C. Cunningham et al.

Table 7.2 Summary of studies investigating the effect of PRP on


bone healing of the foot and ankle
Author Results
[Ref] Year Treatment Model
Basic science
Oprea 2003 PRP with Rat bone Enhanced
et al. [36] fibrin marrow cells osteogenic cell
overlay recruitment and
proliferation
Kanno 2005 PRP Human Increased mRNA
et al. [37] osteosarcoma expression
cells of osteogenic
markers
Gandhi 2006 Local Rat femur 4-fold increase of
et al. [40] percutaneous fracture local growth factor
PRP levels, improved
time to union
Nather 2012 PRP with Rabbit tibial Improved time to
et al. [41] allograft defect union
Clinical applications
Gandhi 2003 PRP with Foot and Resolution of
et al. [44] autograft ankle non- non-union
union
Bibbo 2005 PRP or PRP High risk Improved time
et al. [45] with graft foot and to union, fewer
ankle surgery complications
Barrow 2005 PRP with Syndesmotic Improved time to
et al. [46] autograft fusion union, lower non-
union rate
Coetzee 2005 PRP with Syndesmotic Improved time to
et al. [47] autograft fusion union, lower non-
union rate
Wei et al. 2012 PRP with Displaced Improved time to
[48] allograft type II union, outcome
calcaneal matched that of
fractures autograft alone
Chapter 7. Platelet Rich Plasma 155

Carafino et al. studied the effect of PRP on human tendon


cells when administered with corticosteroids or local anes-
thetics, both common adjunctive treatments for tendinopathy
[39]. They compared the use of two different preparation
methods of PRP (single spin vs. double spin) against a nega-
tive control (no cell culture media) and a positive control
(10 % fetal bovine serum). Both PRP preparations stimu-
lated significantly greater tenocyte proliferation than both
controls, with no significant difference between the two PRP
preparations themselves. Proliferation of tendon cells was
also significantly inhibited in the presence of lidocaine, bupi-
vacaine, and methylpredinose when compared to control
groups, with methylprednisone having the least inhibitory
effect on tenocyte proliferation. These agents should there-
fore be used carefully in patients receiving PRP therapy, as
they may counteract its beneficial effects on healing.
Animal models have also investigated the effect of PRP on
osseous and soft tissue healing. Gandhi et al. used a diabetic
rat model to investigate the effect of percutaneous PRP treat-
ment on fracture healing [40]. Growth factor levels were sig-
nificantly decreased in the non-diabetic control group, while
activated PRP resulted in a fourfold increase. PRP treatment
also normalized cell proliferation and mechanical properties
of the diabetic group to levels not statistically different from
the non-diabetic group. These results illustrate the function of
PRP in enhancing local growth factor levels, and also suggest
a role for PRP in patient populations at high risk for non-
union or wound complications, such as in diabetes. Nather
et al. studied the effect of PRP-augmented allografts in rabbit
tibial defects [41]. At 12 weeks post-surgery, bone bridging
occurred at the host-graft junction in both the autograft
treated group and the PRP-augmented allograft group, while
the allograft alone group did not show bridging until 24
weeks. PRP helped to incorporate the allograft and decreased
the time to union.
Chen et al. recently investigated the use of PRP in the rat
Achilles tendon [42]. Tendons were either treated with botu-
lism toxin to produce mechanically unloading or left
156 C. Cunningham et al.

untreated to produce loading. The tendons were then treated


with either phosphate buffer solution (PBS), tendon stem
cells (TSC), PRP, or a combination of TSC and PRP. Loaded
tendons treated for 14 days exhibited higher collagen I
mRNA expression and collagen III protein levels in all
loaded groups when compared to the unloaded groups.
Additionally, increased collagen III was found in unloaded
cells treated with both TSC and PRP when compared to
unloaded cells of all other groups, suggesting a synergistic
effect. These finding suggest not only a role for PRP treat-
ment, but also that tendon repair is better achieved in loaded
conditions, such as an exercise regimen. Similarly, Mifune
et al. demonstrated that, when added to muscle-derived stem
cells (MDSC), PRP significantly improved articular cartilage
repair at 4 weeks after treatment when compared to MDSC
alone [43]. The addition of PRP increased collagen synthesis,
suppressed chondrocyte apoptosis, and enhanced the inte-
gration of the transplanted cells, which supports the results
produced by Kruger et al [38].

Clinical Applications

One of the first reports of PRP use in the foot and ankle is by
Gandhi et al. [44], who studied 9 patients diagnosed with foot
or ankle fracture non-unions. Initial surgery occurred within
20 days of injury, and the diagnosis of non-union was formu-
lated 4–10 months later. Revision surgery consisted of PRP
and autologous bone graft applied locally to the site of non-
union. Ultimately, union was achieved in all patients within
60 days. Additionally, although plasma levels of PDGF and
TGF-b were consistent between groups, the levels in fracture
hematoma were significantly reduced between patients with
non-union compared to those with an acute fracture. The
authors concluded that the addition of PRP provided critical
early growth factors locally to the site of nonunion to aid the
healing process.
Chapter 7. Platelet Rich Plasma 157

Bibbo et al. used PRP in 62 patients at high risk of delayed


or non-union in the foot and ankle [45]. High risk patients
included active smokers, diabetics, immunocompromised
patients, clinical history of poor bone healing, multiple sur-
geries at the proposed surgical site, and a history of trauma or
infection at the site (Table 7.3). Over 120 different operative
procedures were employed, and patients were followed for 6
months after receiving PRP at the surgical site. Some patients
also received a bone graft at the time of surgery in addition
to PRP. A 91 % union rate was achieved at an average of 41
days. Limitations to this study include the treatment of vary-
ing pathologies, use of different surgical techniques, and the
absence of a control group.
The effect of PRP therapy on syndesmotic fusion with the
use of the Agility total ankle replacement (TAR) has also
been studied. Barrow et al. followed 20 patients undergoing
TAR who received PRP-augmented bone grafts for syndes-
motic fusion [46]. PRP was sprayed onto bone and prosthetic
surfaces, as well as mixed into the graft and packed into the
joint. Compared to the historical rate of 62–82 % fusion rates,
this study found 85 % fused at 2 months, 95 % at 3 months
and 100 % at 6 months. Likewise, Coetzee et al. compared
fusion rates of 66 patients compared to 114 historical controls
[47]. Their results supported those of Barrow et al, showing
statistically significant improvement in fusion rates at 8 and
12 weeks from 61.4 % and 73.6 % in controls to 76 % and
93.9 %, respectively, in the treatment group. In addition, a
significant reduction in delayed or nonunion from 15 % to
3–6 % was seen at 6 months in PRP-augmented patients.
Wei et al. studied all displaced type II calcaneal fractures
in their department over a 7 year period [48]. A total of 276
fractures were randomly divided into three treatment groups
which included autograft alone, allograft alone, and PRP-
augmented allograft. After 1 year, all fractures had success-
fully healed but no significant differences were seen between
groups. However, at 2 and 3 years after surgery, both auto-
graft alone and PRP-augemented allograft had significantly
158 C. Cunningham et al.

Table 7.3 Summary of Smoking


risk factors for impaired
osseous healing Diabetes Mellitus
Extremes of age
Corticosteroid use
Immunosuppression
Peripheral vascular disease
History of or active infection
Multiple (>2) surgeries at site
History of open, high energy
trauma at site
Nonunion or pseudoarthrosis at
site

fewer complications and better radiographic outcomes (mea-


sured by Bohler’s angle, angle of Gissane, and dimensions of
the calcaneal body) than allograft alone. However, no differ-
ence existed between groups in clinical outcome measures
such as degree of residual pain, ability to ambulate, range of
subtalar movement, or alignment of the ankle with the
hindfoot.
The use of PRP in soft tissue injuries of the foot and ankle
has also been explored (Table 7.4). Sanchez et al. followed 6
athletes who sustained acute Achilles tendon ruptures [49].
The patients underwent open repair of the tendon with PRP
injected at the repair site and covered with a fibrin scaffold.
When compared to 6 historical controls matched for age, sex
and activity level, the authors found that those treated with
PRP had no wound complications, regained range of motion
earlier, and resumed training activities earlier when com-
pared to those treated with open repair alone. In contrast,
Schepull et al. followed 30 patients with acute Achilles ten-
don rupture who underwent open repair with PRP injection
and found no difference in mechanical variables, transverse
area, or functional outcome compared to the control group
Chapter 7. Platelet Rich Plasma 159

after a year [50]. However, Schepull et al. did not use a fibrin
scaffold with PRP injections, raising the question of whether
its use significantly augmented the performance of PRP
therapy in the previous study.
The results of PRP use in chronic Achilles tendinopathy
have also been mixed. Gaweda et al. followed 14 patients
who received PRP injections following failure of other treat-
ment modalities [51]. They found a statistically significant
increase in functional outcome at 18 months follow-up as
well as improved tendon characteristics by ultrasound exam-
ination. At final evaluation, 2 patients reported persistent
functional limitations; their pain, however, was reduced from
initial presentation. Delos et al. and Monto et al. performed
similar studies that followed patients with chronic Achilles
tendinopathy that failed 6 months of conservative treatment
[52, 53]. Patients were treated with a single ultrasound
guided injection of PRP, and both studies reported signifi-
cant clinical improvement in most patients, and concluded
that PRP could be used safely and effectively to treat chronic
Achilles tendinosis. Sanchez et al. also showed that PRP was
able to improve patient symptoms and functionality when
used to treat post-surgical Achilles tendon complications
[54]. Conversely, in a level I study de Vos et al. found no sig-
nificant improvement in pain, activity, tendinous structural
organization, or degree of neovascularization compared to
placebo after treating 54 patients with a single ultrasound-
guided PRP injection [55, 56]. Similarly, Owens et al. reviewed
all patients treated with PRP injection for chronic Achilles
tendinopathy and found only modest improvement in func-
tional outcome measures with no changes in MRI appear-
ance of the diseased tendon [57].
Plantar fasciitis is also common in athletes, and several
recent studies have attempted to elucidate the role of PRP in
its treatment. Martinelli et al. treated 14 consecutive patients
with chronic plantar fasciitis with PRP injections at three dif-
ferent puncture sites [58]. Excellent results were reported in
9 of 12 patients, and pain scores were significantly reduced.
They concluded that PRP was safe and effective to use for the
Table 7.4 Summary of studies investigating the effect of PRP on soft tissue healing of the foot and ankle
160

Author [Ref] Year Treatment Model Results


Basic science
Carafino et al. 2003 PRP or PRP with Human tendon Increased cell proliferation, steroids and
[39] steroid/anesthetic cells anesthetics inhibit this effect
Chen et al. [42] 2005 PRP or PRP with stem Rat Achilles Increased osteogenic marker
cells tendon expression,
synergistic with stem cells
C. Cunningham et al.

Clinical applications
Sanchez et al. 2007 Open repair with PRP Acute Achilles Fewer complications, quicker return to
[49] and fibrin scaffold rupture normal
activities
Sanchez et al. 2009 Debridement followed Post-surgical Improved symptoms and functional
[54] with PRP complications outcome of both patients
Schepull et al. 2011 Open repair with PRP Acute Achilles No difference in mechanical variables
[50] injection rupture or
functional outcome
Gaweda et al. 2010 PRP injection Chronic Achilles Improved pain and tendon
[51] tendinopathy characteristics on
ultrasound
Delos et al. 2011 PRP injection Chronic Achilles Improved pain and tendon
[52] tendinopathy characteristics on
ultrasound
Montos et al. 2012 PRP injection Chronic Achilles Improved pain and tendon
[53] tendinopathy characteristics on
ultrasound
de Vos et al. 2011 PRP injection Chronic Achilles No change in tendon structure or
[55] tendinopathy vascularity on
ultrasound
de Vos et al. 2010 PRP injection Chronic Achilles No change in pain or functional level
[56] tendinopathy
Chapter 7.

Owens et al. 2011 PRP injection Chronic Achilles Modest improvement in function, no
[57] tendinopathy MRI changes
Martinelli et al. 2012 PRP injection Plantar fasciitis Decreased pain level
[58]
Ragab et al. 2012 PRP injection Plantar fasciitis Improved pain, decreased fascia
[59] thickness and
Platelet Rich Plasma

signal on ultrasound
Aksahin et al. 2012 PRP or corticosteroid Plantar fasciitis Improved pain, superior to
[60] injection corticosteroids
161
162 C. Cunningham et al.

treatment of this condition. This study, however, is limited by


lack of a control treatment group, such as a plantar stretching
group. Similarly, Rabag et al. treated 25 patients with chronic
plantar fasciitis with PRP injection and found 23 out of 25
patients had minimal to no functional limitations and signifi-
cantly reduced pain [59]. Ultrasonography evidenced signifi-
cant changes in fascial thickness and signal intensity after
PRP injection. Aksahin et al. examined this further and com-
pared PRP treatment to corticosteroid treatment of chronic
plantar fasciitis [60]. Thirty patients were treated with a meth-
ylprednisone/prilocaine injection while another thirty were
treated with a PRP injection following prilocaine injection.
Pain scores in both groups were significantly reduced from
6.2 to 3.4 and 7.33 to 3.93 in the steroid and PRP groups,
respectively. When taking into consideration the potential
complications of corticosteroid treatment such as spontane-
ous rupture, as well as the findings of Carafino et al [39]., PRP
may provide a safer alternative.
The use of PRP in the management of osteoarthiritis or
osteochonral lesions of the foot and ankle has not been
extensively studied (Table 7.5). One study by Mei-Dan et al.
recently investigated the effectiveness of PRP versus hyal-
uronic acid (HA) treatment for pain associated with osteo-
chondral lesions of the talus [61]. A total of 30 patients were
included, half in the PRP treatment group and the other half
in the HA treatment group. Three consecutive intra-articular
injections were administered and patients were followed for
6 months. Both PRP and HA treatment groups reported
increased function and decreased pain compared to controls,
with scores significantly more improved in PRP treatment
groups compared to HA treatment groups.

Conclusion
Despite the encouraging results published by some foot and
ankle investigators, the authors of a recent meta-analysis of
studies comparing PRP with control therapy in orthopedic
Chapter 7. Platelet Rich Plasma 163

bone and soft tissue injuries concluded that, given the lack of
standardization of study protocols, platelet separation tech-
niques, and outcome measures, there is uncertainty about the
evidence to support the increasing clinical use of platelet-rich
plasma [62]. Four studies relevant to foot and ankle applica-
tions were included, and all four were found to have a “very
low” quality of evidence. Inconsistent results are also seen
when using PRP as an adjunct in the treatment of conditions
in other areas of the body. For example, the effectiveness of
PRP in foot and ankle applications differ from those reported
for the spine or maxillofacial conditions. This may be attrib-
uted to the different biological, physiological, and mechanical
factors which influence the local milieu for healing in differ-
ent anatomic areas. Better clinical study design in the form of
well-designed, prospective, randomized, clinical studies are
needed to better evaluate the efficacy of PRP therapy.
To date, no standardized method of preparation has been
adopted. In general, a two-step centrifugation technique is
used to separate various components of anticoagulated
blood. The initial “soft-spin” separates plasma and platelets
from red and white blood cells, while the second “hard spin”
separates the platelet-poor plasma. In some preparation tech-
niques, thrombin is added for exogenous platelet activation
facilitating immediate high-dose delivery of critical growth
factors [63, 64]. Calcium chloride may also be used to precipi-
tate fibrin, which acts not only as a scaffold but also as a res-
ervoir for sustained growth factor release [65]. Variations in
preparation techniques including the initial volume of blood
and centrifugation times yield differences in final PRP vol-
umes and platelet concentrations, and therefore the level of
growth factors delivered to the injury site are inconsistent.
Even when specific protocols are used, the potential for
inconsistency exists because of the variability in an individu-
al’s own platelet count and growth factor levels. Such varia-
tions may influence the efficacy of PRP treatment.
Several investigators have examined the difference in con-
tent and concentration of platelets and growth factors among
commercially available preparation devices. Differences in
164 C. Cunningham et al.

Table 7.5 Summary of studies investigating the effect of PRP on


articular cartilage healing of the foot and ankle
Author Results
[Ref] Year Treatment Model
Basic science
Kruger 2003 PRP Human Induced
et al. alone subcortical chondrogenic
[38] progenitor differentiation
cells
Mifune 2005 PRP with Chemically- Increased
et al. stem cells induced collagen
[43] osteroarthritic synthesis,
rats decreased
apoptosis,
enhanced
integration of
transplanted
cells
Clinical applications
Mei- 2012 PRP or Osteochondral Increased
Dan hyaluronic lesions of the function and
et al. acid (HA) talus decreased
[61] injection pain, superior
to HA

platelet count, growth factor levels, and leukocyte count were


discovered. Variations were also found within multiple sam-
ples using the same preparation technique [66, 67]. Different
preparation techniques also resulted in changes in the physi-
ologic appearance of the platelets and amount of cellular
debris in the sample [68, 69]. While age and gender had no
effect on the level of growth factors, women tended to have
higher platelet counts than men [67]. In addition, multiple
blood draws from the same patient may produce wide fluc-
tuations [70]. Further complicating this matter is that an ideal
platelet count for optimal PRP performance has yet to be
Chapter 7. Platelet Rich Plasma 165

defined. Although a dose-dependent relationship exists


between platelet concentration and growth factor levels,
reports are conflicting on whether there is a linear correla-
tion [15, 16].
Several studies have also reported that leukocyte-rich
PRP (LR-PRP) contains greater levels of growth factors
than leukocyte-poor PRP (LP-PRP) preparations [71–73].
In addition to increased levels of growth factors, some argue
that an increased leukocyte concentration may also have
the added benefit of producing an environment less prone
to infection. However, neutrophils have shown to have a
detrimental effect on muscle [74]. In addition, catabolic
gene expression correlates with increased leukocyte con-
centrations, and the presence of inflammatory cytokines
may cause pain when PRP is injected into soft tissue [75].
Recent studies have attempted to characterize the effect of
LR-PRP versus LP-PRP. Dragoo et al. found that, while
LR-PRP produced a greater inflammatory response at 5
days after injection in rabbit patellar tendon, no difference
was observed between groups at 14 days after treatment
[76]. McCarrel et al. found that LR-PRP resulted in increased
expression of inflammatory cytokines in equine tendons, an
effect that was not counteracted by increasing the platelet
concentration relative to the leukocyte count [77]. The
authors concluded that, because of scar tissue formation
from persistent inflammation, LP-PRP may be superior for
tendon healing.
Mazzocca et al demonstrated that three different prepa-
rations of PRP had differing effects on target cells [78]. The
first method yielded a PRP product with a lower platelet
concentration relative to the other two. Treatment of cells
with this PRP preparation significantly increased cell pro-
liferation when added to osteocytes, myocytes, and teno-
cytes. The second method yielded a LP-PRP, which
significantly increased osteoblast and tenocyte cell prolif-
eration; no significant effect was seen with myocytes. The
LR-PRP preparation significantly increased tenocyte cell
166 C. Cunningham et al.

proliferation and decreased osteocyte proliferation com-


pared to controls but had no effect on myocytes. More
extensive research needs to be performed before these
findings may be applied to the clinical setting. The defini-
tive role of exogenous platelet activation by thrombin on
the efficacy of PRP therapy also remains unclear. Although
a number of preparation systems include its use to facilitate
immediate delivery of growth factors, given the short half
life of these factors, the healing response may be negatively
impacted because of reduced factor availability for later
stages of healing. Additionally, animal studies have demon-
strated decreased ability to induce bone formation with
thrombin-activated PRP [63, 79, 80].
Thrombin also catalyzes the conversion of fibrinogen to
fibrin, which serves as a provisional scaffold during the initial
stages of healing. Fibrin can also bind growth factors, serving
as a reservoir to promote their sustained release [65]. With
such effects, the addition of thrombin or calcium chloride to
PRP to precipitate fibrin is of particular interest. The poten-
tial clinical benefit of enhancing PRP therapy must be further
investigated.
Despite the potential benefits that PRP therapy has dem-
onstrated in basic science studies, clinical findings with
respect to bone and soft-tissue injury have been inconsistent.
Given the paucity of clinical data and lack of standardization
in study design and PRP preparation, the ability to accurately
compare and contrast such data remains difficult. This issue is
critical to determine whether PRP therapy truly enhances the
healing capacity of patients suffering from acute or chronic
foot and ankle conditions. Future research must also attempt
to identify a standardized method of PRP preparation and
the optimal concentration of platelets needed for treatment.
The role of leukocytes, thrombin, and calcium chloride within
these preparations must be further investigated in order to
determine which adjuncts to preparation are beneficial.
Focusing on these issues will help gain more insight into the
application of PRP and its effectiveness in foot and ankle
surgery.
Chapter 7. Platelet Rich Plasma 167

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Chapter 7. Platelet Rich Plasma 173

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Chapter 8
Platelet Rich Plasma
for Biological Therapy:
Applications and Limits
Giuliana Gobbi and Marco Vitale

Introduction
Platelets are not only the primary effectors of hemostasis, but
also carriers of a variety of biologically active factors that can
turn useful for therapeutic purposes, especially, although not
exclusively, tissue healing. Recently characterized platelet-
derived growth factors are able to favor tissue regeneration,
wound healing and angiogenesis. The current availability of
commercial methods to prepare Platelet Rich Plasma (PRP)
has made its use quite popular, although there are still several
areas of uncertainty on the biological efficacy of the prepara-
tions and even on their exact composition, making it essential
to expand our knowledge on the events that characterize

G. Gobbi, PhD
Department of Biomedical, Biotechnological & Translational
Sciences (S.Bi.Bi.T.), University of Parma, Parma, Italy
M. Vitale, MD ()
Anatomy & Histology Unit, Department of Biomedical,
Biotechnological & Translational Sciences (S.Bi.Bi.T.),
University of Parma, Parma, Italy
e-mail: marco.vitale@unipr.it

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 175


Practice, DOI 10.1007/978-1-4471-7271-0_8,
© Springer-Verlag London 2016
176 G. Gobbi and M. Vitale

PRP action at least to standardize the methodologies.


Although in some case-control studies and in several non-
controlled clinical trials PRP has been found effective, stud-
ies on the efficacy of PRP in human subjects are not yet
sufficient, probably because of its relatively recent clinical
application. The results of most studies are hampered by rel-
evant confounding variables such as the variability of PRP
characteristics even in patients with similar platelet counts.
We focus on the sate-of-the-art in this promising field.

Molecular Biology
PRP, defined by at least 1,000,000 platelets/µl in 5 ml of plasma
[1] and by a full array of clotting and growth factors [2], acts as
a biologically active factors reservoir, that induce mitogenesis,
chemotaxis and angiogenesis at the site of application [1, 3, 4].
However, several platelet-enriched products are collectively
named PRP, even though many differences in composition and
preparation techniques have been described, confounding the
evidence-based positive effects of “PRP” therapy.

PRP Preparation

PRP preparations were originally developed in blood trans-


fusion units to be used first for hemostasis during surgical
operations [5] and subsequently, for wound treatments [6].
Although several PRP-based products are currently in use,
the main feature of PRP is its autologous nature. Clinical
efficacy of PRP can be expected at platelet concentrations
four to sixfolds higher than whole blood [3, 7], carrying a
supraphysiological content of autologous growth factors.
Several procedures can be applied for PRP preparation
and, to date, a unique definition of PRP and a unique prepa-
ration protocol do not exist. Typically, active PRP is prepared
immediately before use by a simple 2-step procedure
(Fig. 8.1). Just to begin, each single step is controversial. Step
8 Platelet Rich Plasma for Biological Therapy 177

1: two centrifugations, the first designed to eliminate red


blood cells, the second to enrich plasma with platelets (mini-
mum 1 × 106/μl to obtain clinical efficacy). Step 2: activation of
platelets by the addition of thrombin or calcium chloride,
resulting in fibrin polymerization and production of a gelati-
nous platelet gel applied to the affected site. Indeed, some
Authors suggest to avoid either double centrifugation (Step
1) or platelet activation (Step 2). Leukocytes can be included
in PRP in different proportions, but there are not sufficient
data available to demonstrate functional differences between
leukocyte-rich and leukocyte-poor PRP, even though some
local antibacterial effects can be assumed in the former

FIRST STEP SECOND STEP

Thrombin or
Calcium Chloride

A1 A2 B1 B2
C2

Application at
site of wound

C1

Injection at site
of wound

Fig. 8.1 Preparation of PRP. Active PRP is prepared immediately


before use by a 2-step procedure. Step 1: two centrifugations, the first
(A1) designed to eliminate red blood cells, the second (A2) to enrich
plasma with platelets (minimum 1 × 106/μl to get clinical efficacy).
Step 2: Plasma rich in platelets (B1) can be used immediately (C1)
or activated with the addition of thrombin or calcium chloride (B2),
resulting in fibrin polymerization and production of a gelatinous
platelet gel applied to the surgical site (C2)
178 G. Gobbi and M. Vitale

instance. According to some authors, leucocytes should be


discarded from the preparation to prevent inflammatory pro-
cesses [8]. However, most of the PRP used contain leucocytes,
and there is no good scientific reasons to discard them [9].
The several products on the market to obtain autologous PRP
can differ from each other in the preparation procedure and
results. Different systems, in fact, have different yields in terms of
concentrated viable platelets, with these differences accounting
for many of the criticisms regarding the efficacy of PRP [10].
In brief, PRP may include leukocytes or not and/or a vari-
able platelet enrichment; protocols may involve the use of
platelet agonists (i.e. thrombin or calcium), to form specific
scaffolds, and/or the combination with gelatin hydrogels, to
control growth factors release [11] (Fig. 8.2).

PB PRP NOTHING

AGONISTS

FIBRIN

COLLAGEN
PRP
LY
ON
HYDROGEL

P
BIOLOGICAL GRAFT
LE
UK RP & (i.e. DBM)
OC
YT
ES

SYNTHETIC GRAFT
(i.e. HA)

CELLS
(i.e. MSC; BMSC;
BM L-PRP CHONDROCYTES)

Fig. 8.2 Different PRP-derivative products. Primary source for PRP


preparations can be represented by both peripheral blood (PB) and
bone marrow (BM). Consequently, PRP contain only platelet-rich
plasma (PRP) or also leukocytes (L-PRP). Before clinical or exper-
imental use, both PRP and L-PRP might be combined with biologi-
cal factors, scaffolds, cells or association of two or more of these.
Almost all the resulting products are commonly named as “PRP”.
DBM demineralized bone matrix, HA hydroxyapatite, MSC mesen-
chymal stem cells, BMSC bone marrow stem cells
8 Platelet Rich Plasma for Biological Therapy 179

Main Cell Populations Trapped in PRPs

Based on the PRP production technique, it is possible to


entrap in the nascent gel several other cell populations,
although their effects on the clinical efficacy of PRP have not
been well clarified. We recently developed a rapid method to
obtain mesenchymal stem cells and platelets, starting from
bone marrow aspirates. In parallel to platelet enrichment, we
recovered also mononuclear cell, such as CD3pos T cells, and
mesenchymal cells, which preserved their osteogenic, adipo-
genic and chondrogenic potential [12] with demonstrated
advantages for bone repair. Despite the various possible cel-
lular components in PRP preparations, the functionally rele-
vant cell populations remain platelets and leucocytes.
Platelets originate from precursors, named proplatelets,
strings of discoid structures containing granules (Fig. 8.3).

a b c

Fig. 8.3 Platelet precursor. Platelets origin from long extended pre-
cursors, named proplatelet, similar to strings of discoid structures
containing granules. Granules are distributed in platelet cytoplasm
(a). Platelet surface expresses several glycoproteins (GP), mediators
of platelet adhesion to the extracellular component. GPIIbIIIa (b)
represents the fibrinogen receptor, one of the most expressed pro-
teins on platelet surface. The merged image (c) shows the distribu-
tion of granules and surface receptor
180 G. Gobbi and M. Vitale

Proplatelets derive from megakaryocytes (MKs), from which


they inherit cytoplasmic granules [13]. Mature platelets are
anucleated cells functionally characterized as primary
effectors of hemostasis containing many granules of different
types. Granules are storage pools of active substances released
following adhesion to extracellular matrix components, such
as collagen, or in response to soluble agonists. There are three
main types of platelet granules: dense (or delta), alpha gran-
ules and lysosomes.
Dense granules contain several active substances, includ-
ing ADP/ATP, serotonin and Ca2+. ADP stimulates platelet
activation and hemostasis [14]; serotonin mediates vasocon-
striction and capillary permeability [15], while calcium is
essential for fibrin formation [16]. Alpha-granules are pack-
ages of growth factors, chemokines and cytokines [17, 18].
Platelet lysosomes contain enzymes active on protein and
matrix degradation [19].
Platelets play a key role in the healing process, by the
activation-induced release of alpha-granules, secreting more
than three hundreds proteins [20] able to induce mitogenesis,
chemotaxis and angiogenesis at the site of application.
Leukocytes, producing growth factors as well, can act in
synergy with platelets providing an additional source of bio-
active proteins [21] and a primary line of defense for infec-
tion [22]. Leukocytes classically roll and adhere to inflamed
endothelium, or can be recruited by platelets at the site of
vessel injury. Indeed, the platelet-leukocyte crosstalk is gain-
ing attention in several processes, such as immunity and tis-
sue healing [23–26].

Chemokines, Cytokines & Growth Factors

Tissue repair and regeneration, as well as removal of necrotic


tissues, are mediated by several factors, stored in platelets
and leukocytes, able to regulate angiogenesis and to recruit
macrophages, mesenchymal stem cells, and osteoblasts
(Fig. 8.4).
8 Platelet Rich Plasma for Biological Therapy 181

PRO-ANGIOGENIC ANTI-ANGIOGENIC
ACTIVITY ACTIVITY
PDGF-AA, -BB, -AB PF4
FGF Angiostatin
HGF Endostatin
VEGF TIMP-1, -4
EGF TSP-1
IGF-1 TGF-beta

PLATELET
BIOACTIVE FACTORS ARE
STORED IN PLATELET AND
RELEASED UPON
ACTIVATION

CHEMOTAXIS ACTIVITY
TGF-beta; PDGF-AA, -BB, -AB; FGF; HGF; VEGF; EGF;
IGF-1; BMP-2, -4, 6; CCL5; CCL3; CXCL-4, -7, -12

Fig. 8.4 Principal PRP bioactive factors involved in angiogenesis


and chemotaxis. Platelet granules contain storage pools of active
substances released upon activation, following adhesion to extracel-
lular matrix components such as collagen or in response to soluble
agonists. Indeed, platelets represent a reservoir of bioactive factors
able to regulate angiogenesis, to recruit blood cells, to induce mito-
genesis and cell differentiation. See the text for fully explanation

The major platelet chemokines are CXCL7, also named


beta thromboglobulin (beta-TG), CXCL4 (platelet factor 4 –
PF4) and CCL5 (also known as RANTES) [27, 28]. These
molecules, stored altogether in alpha-granules [29], are
released upon platelet activation, acting as chemoattractants
for leukocytes and modulator for platelet aggregation, angio-
genesis and immunity [27]. Indeed, another well-characterized
platelet-stored chemokine is CXCL12, also known as
SDF1alpha, regulating bone marrow and hematopoietic
homeostasis and neovascolarization [30, 31].
Regeneration after vascular injury [32–34] and recruit-
ment of monocytes and neutrophils to the vascular wall are
induced by released chemokines, while interleukin (IL)
-1beta, -6 and -8 and other secreted cytokines induce inflam-
matory responses in endothelial cells [35, 36].
182 G. Gobbi and M. Vitale

Platelet-derived growth factor (PDGF)-A and B (the two


predominant forms of PDGF in platelets), vascular endothe-
lial growth factor (VEGF), insulin-like growth factor (IGF)
-1, fibroblast growth factor (FGF), hepatocyte growth factor
(HGF) and epidermal growth factor (EGF) have a pro-
angiogenic activity, and their secretion can be promoted by
CD40L (CD154)/CD40 interaction [37]. Platelet-released
soluble CD154 induces the production of IL-6 and tissue fac-
tor (TF) as well as B cell differentiation, adaptive immune
responses [24], endothelial cell proliferation and migration
and activation of angiogenesis [38].
Platelets contain pro- and anti-angiogenic factors at the
same time, stored in distinct subsets of alfa granules [39]. The
secretion of these factors is differentially regulated by the
selective engagement of the thrombin receptors PAR-1 and
PAR-4 [40]. The activity of pro-angiogenic factors secreted by
platelets accounts for increased vessel permeability, leuko-
cyte rolling on endothelial cells [41], vascular cell migration
and proliferation, as well as sprouting of new vessels [42] at
the site of injury.
Anti-angiogenic factors include transforming growth
factor (TGF)–beta1 [43] and thrombospondin (TSP)-1 [44],
Platelet factor 4 (PF4), angiostatin, endostatin and tissue
inhibitor of metalloproteinases (TIMP)-1 and -4 [45, 46].
On these basis, it has been suggested that also pro- and
anti-inflammatory factors might be selectively packaged in
different subsets of alpha-granules [24]. Alpha-granules
also contain growth factors that favor osteoclastogenesis,
such as IL-1 and macrophage inflammatory protein (MIP)-
1alpha, also known as CCL3 [47–49]. Insulin-like growth
factor (IGF)-1 stimulates bone matrix formation and pro-
liferation of osteoblasts and their precursors [50]. MKs
synthesize bone morphogenetic proteins (BMPs)-2, -4 and
-6 which are released by platelets and are essential for bone
formation [51].
Finally, in addition to this well-known activity of release of
granule-stored molecules, platelets are also able to up-take
circulating factors, regulating their blood levels.
8 Platelet Rich Plasma for Biological Therapy 183

Mechanisms and Timing of Factors Release

The platelets present in PRP function as a tissue sealant ini-


tiating wound repair [52], while fibrin matrix acts as drug
delivery system slowly releasing platelet-derived bioactive
factors [3].
The release of growth factors starts about 10 min after
platelet clotting, and most growth factors are released within
one hour, even though additional growth factors may be syn-
thesized and released for a further 7 days [10, 53].
Mazzucco et al. [54] demonstrated that the kinetics of
growth factor release can be influenced by the method used
to obtain PRP. Consistent variations between different prep-
aration methodologies were found in the concentration and
release of PDGF-BB, TGF-b1, b-FGF, VEGF, EGF and
IGF-I and BMP-2 measured at 20 min, 1 h, and 7 days after
gel formation. Interestingly, some groups [55] showed no cor-
relation between platelet and growth factor concentration in
PRP, while, on the contrary, others did [52]. Of note, the
growth factor yield and release kinetics from a gel can be
very different, even when similar methods for platelet gel
preparation are used. Several factors may account for this
lack of uniformity, including a variable susceptibility of plate-
lets to manipulation-induced stress, microaggregates impair-
ing platelet counts, and presence of growth factor-binding
proteins [56].

Mechanism of Wound Healing

Wound healing is a complex process involving several cell


types and bioactive factors. A simple model is based on 3
main steps: inflammation, cell proliferation and tissue remod-
eling. Although several different cytokines and growth fac-
tors characterize each step with some specificity, the whole
process is rather a continuum of events mutually influenced
in terms of promotion and inhibition of their progression
[26]. Following tissue injury, platelets are recruited at the site
184 G. Gobbi and M. Vitale

of damage, activated and induced to release the bioactive fac-


tors stored in their granules. Serotonin and histamine from
dense granules increase capillary permeability to facilitate
the recruitment and activation of inflammatory cells at the
wound site, while clotted platelets provide an ideal surface
for leukocyte adhesion [57].
As a consequence of platelet activation, several mitogenic
factors, such as PDGF, act on fibroblast and other cells pro-
moting proliferation and the so-called granulation tissue.
Fibroblasts can differentiate into myofibroblasts, when
exposed to growth factors, and both these cells can produce
extracellular matrix (ECM) proteins and participate in subse-
quent tissue remodeling [58].
PRP can: (i) inhibit cytokine release from macrophages,
improving tissue healing and regeneration by limiting the
inflammation process; (ii) promote new capillary growth; (iii)
accelerate epithelialization in chronic wounds. PRPs also
have a role in the local non-specific immune response as they
produce signaling proteins that attract macrophages and con-
tain a variable amount of leukocytes which show a definite
antimicrobial activity.

Clinical Applications
The rationale for the therapeutic use of PRP is to make
platelet-derived factors locally available for tissue to be
healed. Platelets present in PRP function as a tissue sealant
initiating wound repair [52], while fibrin matrix acts as drug
delivery system releasing platelet-derived bioactive factors
[3]. Local milieu conditioning modifies precursor cell differ-
entiation and angiogenesis, ultimately leading to tissue regen-
eration [59–61]. Indeed, PRP has gained success particularly
in the context of traumatic injuries of poorly vascularized or
ischemic tissues, where the local availability of autologous
pro-differentiative/pro-angiogenic factors more easily boosts
a positive clinical outcome. Moreover, although not yet
standardized, autologous PRP has less safety concerns than
8 Platelet Rich Plasma for Biological Therapy 185

other cell-based regenerative therapies. The therapeutic use


of PRP was pioneered in dentistry [62]: nowadays, although
more on the basis of empiricism and anecdotal reports than
on a robust scientific knowledge [63, 64], its clinical applica-
tions exploded to other fields, including cardiac surgery [65],
ophthalmology [66], oral and maxillofacial surgery [53], plas-
tic surgery [67, 68], cosmetic medicine [69, 70], sports medi-
cine and orthopedics.
In the orthopedic field, four of the main applications of
PRP refer to: (1) chronic tendinopathies; (2) acute ligamen-
tous injuries; (3) muscle injuries; (4) intraoperative augmen-
tation [58].
In tendinopathies, PRP is usually injected at the site of
injury to accelerate tendon healing and recovery [71].
Treatment is usually followed by a period of rehabilitation
with a gradual return to activities. Several studies focused on
tendinopathies, suggesting a better clinical outcome with
PRP for lateral epicondylitis [72–74] and chronic patellar
tendinopathy [75, 76]. However, de Vos and co. [77] did not
find significant differences between PRP and saline solution
injection in a block-randomized, double-blind, placebo con-
trolled trial on 54 patients affected by chronic mid portion
Achilles tendinopathy.
Professional athletes often experience acute ligamentous
injuries and, as they need quick functional recovery, they are
good candidates for PRP treatment. Nguyen and co. [78] esti-
mated that 1-week PRP treatment could anticipate the return
to sport activity of about 2 weeks in patients with medial col-
lateral ligament injuries. In anterior cruciate ligament inju-
ries, the use of bioactive scaffolds could improve the effects
of PRP products in animal models [79, 80], which, if used
alone, did not produce any significant effect compared to
controls in human [81, 82].
Muscle injuries are frequent in athletes, compromising
their season and demanding innovative therapeutic
approaches to allow a quick return activity. For this reason,
PRP has been introduced in the management of muscle
injuries, suggesting a potential role of platelet-stored growth
186 G. Gobbi and M. Vitale

factors in muscle regeneration [83]. Indeed, as discussed


above, tissue healing is characterized by several steps, ending
with tissue remodeling, each of which requires the interven-
tion of specific chemokines, cytokines and growth factors.
However, to date, no human studies are available supporting
this hypothesis. On the contrary, although in animal models
PRP favors tissue regeneration [84], other published data
suggest a potential role of TGF-beta released by PRP in the
induction of a fibrotic healing [85], increasing the risk of new
injuries.
Arthroscopic rotator cuff repair is the kind of intervention
requiring intraoperative augmentation. However, the use of
PRP to enhance healing in the treatment of injured rotator
cuff is not recommended now. In a level 1 of evidence ran-
domized controlled trial, Castricini and co. [86] did not find
evidence for the use of platelet-rich fibrin matrix for augmen-
tation of a double-row repair of a small or medium rotator
cuff tear. On the contrary, platelet-rich fibrin matrix might
have negative effects on rotator cuff repair [87].
Osteoarthritis and articular cartilage degradation repre-
sent one hot topic in PRP research applications. A large
number of studies are available on the potential effects of
PRP and its derivatives on osteogenesis and chondrogenesis.
However, most studies have been conducted in vitro on pri-
mary human cells or on animal models, and the few human
studies available are not randomized controlled trials.
Nevertheless, Kon et co. [88] reported good results in a pro-
spective cohort study of 115 subjects with knee osteoarthritis
treated with intra-articular injections of PRP. The results
were inversely related to age, body mass index and severity of
damage. On the other hand, studies on animal models pro-
duce satisfactory results. PRP has been administered alone
[89, 90], associated to different scaffolds [89, 91, 92], or in
combination with chondrocytes [93], with osteochondral tis-
sue formation. Similar results were obtained in animals for
the treatment of bone defects, where PRP at the site of frac-
ture improved callus formation and osteogenesis [94, 95]. The
effects of PRP are boosted by biodegradable scaffolds, such
8 Platelet Rich Plasma for Biological Therapy 187

as gelatin hydrogel [25, 96], that do not interfere with the rate
of bone regeneration. On the contrary, the beneficial effects
of PRP associated to grafts are still unclear. Both biological
and synthetic bone grafts support bone healing [97–99] and
the addition of PRP was beneficial in some cases [100–102],
irrelevant in others [103, 104], or even detrimental [105].

Limits
Despite the diffusion of PRP preparations in the treatment of
several injuries, there is a lack of solid knowledge on its real
benefits. This gap is due to several criticisms on the clinical
and experimental approaches to this topic. In a very sche-
matic way, we could summarize PRP flaws as follows.
1. Nomenclature. Indeed, the term PRP embraces a number
of different biological preparations not even well
characterized.
2. Processing. Frequently, details about the techniques used
to obtain PRP and to apply it are not well defined. Indeed,
platelet count is often not reported, as well as the centrifu-
gation step used for platelet enrichment. The amount of
PRP injected or applied is often left to the opinion of the
clinician, as well as the frequency of PRP applications. For
these reasons it is difficult to compare methods and out-
comes, hampering the necessary standardization.
3. Formulation. There is a lack of published information on
the formulation of different PRP-derived products, in
terms of cells and bioactive factors content. The content of
growth factors in platelets is highly variable between indi-
viduals, independently from platelet count. Moreover,
platelets are extremely sensitive to any kind of stress, from
blood drawing to PRP gel production. Thus the amount of
platelet-derived factors available at the end of the manipu-
lation process is likely to depend on the cumulative effects
over platelets, along the entire preparation process.
4. Exploitation. To date, only few randomized controlled tri-
als have been performed in humans to provide the level I
188 G. Gobbi and M. Vitale

evidence for the efficacy of PRP use, and no standardized


administration protocol is available. The majority of pub-
lished data come from small trials, frequently without a
control group. Large, methodologically rigorous, random-
ized controlled trials, using clear primary clinical and bio-
logical endpoints need to be conducted.
Although PRPs represent a formidable tool for clinical
application, many questions are still open, including the
appropriate indications for its clinical use as well as the effec-
tive concentrations and quantities for each product to be
used in each therapeutic situation. Given what has already
been demonstrated and what is promised based on the avail-
able data in terms of benefits for the patients, a special
research effort in the PRP field would be worth its costs.

Economical Issues
Both the cellular and molecular components of PRP are
autologous, as opposed to the use of recombinant human
growth factors, such as PDGF-BB, which have been recently
associated to malignant transformation in human cells [106].
PRP is autologous and, for this reason, safer than allogenic
preparations. It does not induce immunogenic reactions [2],
and disease transmission is not an issue [2, 10, 107].
Given the availability of simple techniques for PRP pro-
duction, PRP is considered an inexpensive way of enhancing
tissue healing, and its use may be a more cost-effective and
economical approach for the required treatments. Indeed,
PRP represents an easy, cost-effective way to obtain high
concentrations of growth factors at the site of application.
However, several devices have been developed and commer-
cialized for clinical preparations that are promoted in the
absence of solid clinical data on the outcome or effectiveness
in tissue repair [108].
Dougherty [109] compared the cost-effectiveness of PRP
in the treating non-healing diabetic foot ulcers to several
other therapies over a 5-year period in an hypothetical group
8 Platelet Rich Plasma for Biological Therapy 189

of 200,000 patients. PRP was the cheaper approach (15,159


USD) and, on the contrary, the use of PDGF-BB was the
most expensive (47,252 USD).

Perspective for the Future


PRP preparations have raised a considerable interest toward
new possibilities for efficient tissue repair and regeneration
and, in principle, PRP has a marked potential in this field.
Being autologous, PRP has less safety concerns than other
cell-based regenerative therapies. Indeed, regenerative treat-
ments might even partially substitute surgical management in
some injuries, reducing the time of recovery.
In this perspective, it will be of interest to understand the
potential synergistic effects of PRP combined with different
scaffolds in tissue regeneration. Despite the intensive research
effort on PRP, open questions are still more numerous than
answers, and further large, well-designed, randomized con-
trolled trials are needed to standardize procedures and con-
vincingly demonstrate the benefits of PRP in terms of clinical
outcome.

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Chapter 9
The Systemic Effects
of Platelet-Rich Plasma
Amy S. Wasterlain, Hillary J. Braun, and Jason L. Dragoo

While PRP was introduced in the 1970s, there has been a


recent geometric increase in its use for sports-related injuries
[28], and it is now used to treat over 86,000 athletes annually
in the United States [26] and many others throughout Europe
[2, 25, 64, 80]. At the same time, the World Anti-Doping
Agency (WADA) has become increasingly concerned that
PRP injections may constitute a doping violation because
PRP contains large quantities of ergogenic growth factors.
Although the contents of the PRP preparations themselves
have been extensively studied [10, 22, 25, 38, 89], the systemic
effects and potential doping implications of PRP therapy are
just beginning to be explored. Similarly, medical concerns
related to the growth factors contained within PRP have
been raised, although the long-term effects of PRP have not
yet been directly addressed.

A.S. Wasterlain, MD • H.J. Braun, MD • J.L. Dragoo, MD ()


Department of Orthopaedic Surgery and Sports Medicine,
Stanford University School of Medicine,
450 Broadway, Redwood City, CA 94063, USA
e-mail: amywasterlain@gmail.com; jdragoo@stanford.edu

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 199


Practice, DOI 10.1007/978-1-4471-7271-0_9,
© Springer-Verlag London 2016
200 A.S. Wasterlain et al.

Doping Concerns with the Use of PRP

The question of whether autologous blood products should


be permitted has been under close scrutiny since a British
sports physician made enquiries to WADA and UK Sport, an
anti-doping organization, in 2005 [15]. The concentration and
reinjection of the patient’s own blood immediately prior to
competition has been prohibited at least since 2005 [94],
because the procedure increases blood oxygen levels and
therefore could artificially enhance performance; this should
not be a concern with PRP, because PRP by definition con-
tains extremely low quantities of red blood cells.
Platelet-derived preparations including PRP were first
regulated by WADA under the 2010 Prohibited List because
of concerns that the elevated concentrations of growth factors
in PRP may confer an unfair advantage to treated athletes.
Regulations for PRP therapy differed by treatment site: intra-
muscular PRP injections were strictly prohibited, while intra-
tendinous PRP was permitted only with a therapeutic use
exemption. The 2010 Prohibited List also banned any mole-
cule affecting muscle, tendon, or ligament protein synthesis/
degradation; vascularization; energy utilization; regenerative
capacity; or fiber type switching because these processes have
known or potential benefits for athletic performance [35].
WADA lifted the ban on PRP in 2011 in recognition of the
lack of evidence to support a systemic performance-enhanc-
ing effect and to allow further research in the field. However,
the aforementioned molecules and many of the growth factors
contained within PRP have remained banned under the
Prohibited Lists [95–97].

Growth Factors
The use of other growth factors in athletes is prohibited
under section S2 of the 2013 WADA prohibited list [97], par-
ticularly because of concerns regarding insulin-like growth
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 201

factor I (IGF-I) and the potential for its abuse as an ergo-


genic substance. PRP contains elevated concentrations of
growth factors and cytokines associated with the α granules
of platelets, including platelet derived growth factor (PDGF),
vascular endothelial growth factor (VEGF), and basic fibro-
blast growth factor (bFGF), all of which may enhance healing
through promoting cell proliferation, chemotaxis, cell differ-
entiation and angiogenesis [37]. Indeed, the same growth
factors that mediate PRP’s putative healing effects are
understandably of concern to WADA, athletes and their phy-
sicians, and all other parties with an interest in promoting
clean and fair competition.
WADA’s prohibition of specific growth factors and related
substances that are associated with PRP reflects the view that
high quantities of these molecules in circulating blood may
be advantageous to athletes. Although PRP may also induce
local changes within the tissues that augment healing and
may lead to local changes in performance of muscle, ergo-
genic effects of PRP would presumably be mediated through
systemic increases in growth factor levels.
Some of the growth factors contained within PRP that
may confer or mediate PRP’s putative systemic effects are
described below, and are summarized in Table 9.1.

Basic Fibroblast Growth Factor (bFGF)

bFGF, otherwise known as FGF-2, contributes to angiogene-


sis by stimulating the proliferation of endothelial cells, and
interacts with TGF-β and PDGF-BB to enhance proliferation
of satellite cells, the stem cells of mature muscle [16]. Muscles
treated with bFGF show improved healing and significantly
increased fast-twitch and tetanus strength [63], and intramus-
cular administration of bFGF induces significant local angio-
genesis in animal muscle [24]. bFGF is also increased in
response to long-term training regimens [45]. Together, these
reports suggest that bFGF may induce muscle hypertrophy
and increase oxygen transport, thereby enhancing athletic
202 A.S. Wasterlain et al.

Table 9.1 Growth factors contained within PRP and/or banned by


WADA
PRP increase Half-life
Growth relative to Banned by
factor blood WADA
bFGF 48× [90] Yes 85 m [83]
HGF Yes 30 m [8]
hGH <1.0× Yes
IGF-I <1.0× Yes 10 m (free) [14, 30]
16 h (IGFBP-3
complex) [14]
IGFBP-3 <1.0× No 30–90 m [103]
PDGF 67× Yes 2 m [13]
(PDGF-BB)
[90]
VEGF 6× [90] Yes 3 m [27]
PRP increase is reported relative to serum (hGH, IGF-I, IGFBP-3,
bFGF, VEGF) or plasma (PDGF) [90]. Banned molecules reported
based on WADA Prohibited List
bFGF basic fibroblast growth factor, HGF hepatocyte growth factor,
hGH human growth hormone, IGF-I insulin-like growth factor I,
IGFBP-3 insulin-like growth factor binding protein 3, PDGF
platelet-derived growth factor, VEGF vascular endothelial growth
factor

performance. bFGF is not an ideal candidate for detection of


doping because it is present in the blood only at very low
concentrations, typically less than 1 ng/mL, and is primarily
localized to skeletal muscle [45].

Hepatocyte Growth Factor (HGF)

HGF is a heparin binding protein released by platelet


degranulation that is mitogenic for endothelial cells and
stimulates fibroblast proliferation [36]. HGF induces
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 203

expression of VEGF, and the combination of HGF and


VEGF synergistically promotes neoangiogenesis [84, 99]. It
is explicitly banned by WADA under Section S2 [97, 98].

Human Growth Hormone (hGH)

Growth hormone has received much attention as a potentially


performance-enhancing substance; it has been dubbed “the
most anabolic substance known,” [74] and has been an impor-
tant target of WADA’s anti-doping campaigns over the past
decade. hGH doping may appear attractive to some athletes
because it is lipolytic, and because supra-physiological levels
strengthen connective tissue, thus mimicking the increase in
strength following exercise [85]. Although hGH has anabolic
effects on muscle, and may enhance performance through
increasing lean body mass [93], it may not improve strength
and may actually decrease exercise capacity [54]. hGH doping
in healthy young adults has been associated with adverse
events including soft tissue edema and fatigue [54].
Supra-physiologic hGH detection presents many chal-
lenges. hGH secretion is pulsatile, and is influenced by stress,
exercise, caloric intake, gender, age, and even time of day,
making it difficult to compare intra- and inter-individual hGH
levels [77]. In recognition of these challenges, WADA adopted
the use of both direct isoform and indirect assays for hGH
detection. In the direct isoform approach, the concentrations
of all molecular isoforms of hGH in circulation are measured,
and the ratio of the 22 kDa monomer to total isoforms of
hGH is compared to an established cut-off. Since recombinant
hGH (rhGH) is comprised solely of the 22 kDa isoform,
whereas naturally secreted hGH is present in several isoforms,
a higher 22 kDa: total isoform ratio is suggestive of hGH dop-
ing. This method was employed at the 2004 Olympic Games in
Athens, and in Turin in 2006. Importantly, supraphysiological
hGH levels secondary to PRP therapy cannot be detected by
this assay, because the hGH contained in PRP is produced
naturally by the patient. Therefore, any hGH-related effects of
204 A.S. Wasterlain et al.

PRP may be evaluated more accurately through indirect mea-


surements of IGF-I, IGF binding proteins, and other related
biological factors (see discussions on IGF-I and IGFBP-3,
below). Circulating hGH has a 30 min half-life, and is detect-
able in serum within 3 h following a dose of GH [51].

Insulin-Like Growth Factor I (IGF-I)

IGF-I promotes cell proliferation, differentiation, and hyper-


trophy of skeletal muscle [70]. IGF-I is produced in the liver
in response to hGH, and is the primary downstream mediator
of the actions of hGH [21]. IGF-I exerts anabolic effects on
skeletal muscle and mediates the growth-promoting effects
of hGH [50, 62, 85, 87]. Among the IGF-related molecules,
IGF-I is the most specific marker of supraphysiological hGH
exposure [20]. Circulating IGF-I is 99 % bound to IGF bind-
ing proteins, primarily IGF binding protein 3 (IGFBP-3). The
remaining 1 % is unbound, or “free,” and is believed to exert
the biological effects upon binding to the IGF-I receptor [47].
Therefore, accurate quantification of IGF-I requires that
both free and dissociable (bound) IGF-I be detected. IGF-I
abuse is probably much less prevalent than hGH abuse
because there is currently no natural source for IGF-I, and
thus any exogenous IGF-I dose would have to be obtained
through recombinant DNA technology. Hypoglycemia is a
potential risk of elevated IGF-I or insulin levels.
Unlike hGH, IGF-I secretion is relatively stable, with mini-
mal intra-individual variability over a 24-h period. Studies of
normal subjects and patients with abnormal hGH secretion
have repeatedly demonstrated that IGF-I levels are indicative
of integrated 24-h hGH secretion [7]; specifically, the percent-
age change in IGF-I levels has been validated as an indirect
assay for supraphysiological hGH [82]. Following administra-
tion of a single dose of hGH, Lee et al. found an initial rise in
IGF-I at 3 h concomitant with peak GH levels, and continuing
increases in IGF-I over at least the next 24 h [51].
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 205

Mechano Growth Factor (MGF)


IGF-I has at least two mRNA splice isoforms: IGF-IEa and
IGF-IEc (sometimes called mechano growth factor, MGF
[56]), which some researchers have suggested may exert
endocrine and autocrine/paracrine effects, respectively [70].
The MGF hypothesis asserts that after muscle injury the
Igf1 gene is first spliced into the IGF-IEc/MGF splice vari-
ant, and subsequently translated and processed to yield
mature IGF-I protein as well as an autonomous C-terminal
MGF peptide [42, 101]. According to the theory, splicing can
be shifted towards the Ea mRNA form to promote myo-
blast differentiation, or towards the Ec/MGF mRNA form
to induce satellite cell activation and proliferation. Although
a synthetic version of the MGF peptide has been shown to
influence committed skeletal muscle progenitor cell prolif-
eration, mediate local tissue repair in a damage-sensitive
manner, and induce muscle hypertrophy [5], it is unclear
whether this peptide is indeed produced from the endoge-
nous Igf1 gene [60].
According to the MGF hypothesis, PRP would be pre-
dicted to contain the circulating, plasma-derived IGF-IEa
peptide, rather than the Ec peptide. If this were the case,
PRP would be unlikely to exert performance-enhancing
effects through IGF-I, because it is the Ec/MGF form of
IGF-I that is believed to be ergogenic. However, prominent
endocrine experts including Dr. Andrew Hoffman [43]
emphasize that extreme caution should be used when
extrapolating mRNA data to protein-level actions, as the
MGF hypothesis does. As mentioned previously, any attempt
to distinguish between the IGF-IEa and Ec/MGF peptides
in vivo is further complicated by insufficient data providing
proof of principle that the IGF-IEc peptide is generated in
vivo. Similarly, the abundance of IGF-IEc/MGF mRNA in
skeletal muscle in vivo has never been shown to exceed or
even approach that of IGF-IEa mRNA, including during the
period of muscle repair when IGF-IEc/MGF mRNA is
206 A.S. Wasterlain et al.

transiently increased [41, 42, 67]. The lack of evidence sup-


porting the existence of endogenous MGF protein and the
difficulty and inconsistency of detecting the different IGF-I
forms in vivo present significant barriers to testing IGF-IEa
and IGF-IEc levels at this time. Furthermore, at the time of
writing there are no commercially available human ELISA
kits for MGF.

IGF Binding Protein Type 3 (IGFBP-3)

IGFBPs are proteins that bind to IGF-I and IGF-II, forming


complexes that increase the circulating half-life and facili-
tate delivery of IGFs to tissues. IGFBP-3 mediates the
transport and controls the bioavailability of IGF-
I. Approximately 99 % of the IGF-I in circulation is bound
to IGF binding proteins – primarily IGFBP-3 – and it is this
storage form of IGF-I that is under hGH control [46]. hGH
and IGF-I regulate its production, and secretion patterns of
IGFBP-3 mimic those of IGF-I. IGFBP-3 is not itself ergo-
genic, but IGF-I/IGFBP-3 ratios provide an indirect test for
hGH doping [49]. Multiple studies have shown that the ratio
of IGF-I to IGFBP-3 increases approximately twofold after
administration of recombinant hGH; this observation is
reflected by the fact that IGF-I shows a dose-dependent
response to hGH, whereas IGFBP-3 does not [49, 88]. In a
double-blind placebo-controlled trial, Kniess et al. also
showed that the products of serum concentrations of IGF-I
and IGFBP-3 were markedly elevated in subjects treated
with hGH [48]. Similarly, the product of IGF-I IGFBP-3 is
an important component of an indirect test for hGH doping
called the “discriminant function” developed by Kniess and
colleagues, and has been used by WADA to detect exoge-
nous hGH doping in athletes. The discriminant function is a
mathematical equation composed of serum IGF-I, IGFBP-
3, and N-terminal propeptide of type III procollagen
(PIIINP) concentrations [48].
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 207

Platelet-Derived Growth Factor Type BB


(PDGF-BB)

PDGF is primarily found and stored in the α-granules of plate-


lets [4]. Upon platelet activation, the dimeric isoform PDGF-BB
(the prototype of the PDGF family) is released from platelets
in its active form and has a half-life of less than 2 min [13, 52].
The most important specific activities of PDGF include angio-
genesis and macrophage activation [39, 72]; chemotaxis for
neutrophils, macrophages, fibroblasts and smooth muscle cells
[61]; and proliferation of fibroblasts [65]. PDGF is particularly
important in the initial stages of wound healing, at which time
it triggers inflammatory healing cascades, inducing the synthe-
sis of other growth factors, such as IGF-I [57]. Animal studies
have shown that PDGF-BB increases the strength of wound
tissue and decreases the time of healing [71], and significantly
increases the load-bearing ability of healed tissue [40].

Vascular Endothelial Growth Factor (VEGF)

VEGF is a powerful stimulator of angiogenesis, building new


vasculature to bring additional extrinsic cells, nutrients, and
growth factors to the site of injury [44, 53, 91]. VEGF can be
induced by hypoxia, and VEGF mRNA is up regulated in
human skeletal muscle in response to a single bout of dynamic
exercise [34, 75]. Further, the angiogenic signal triggered by
VEGF is attenuated when exercise training improves capillar-
ity and oxygen diffusional conductance [76], suggesting that
supraphysiological VEGF could serve as a replacement for
natural exercise and training. In contrast, muscle-specific
VEGF deficiency greatly reduces exercise endurance in mice
[66]. VEGF has a circulating half-life of approximately 3 min,
and is elevated immediately in response to an acute bout of
exercise [18, 27]. VEGF could have particularly great perfor-
mance-enhancing effects if it entered systemic circulation and
208 A.S. Wasterlain et al.

exerted its effects in tissues other than at the site of injury; its
ability to enhance oxygen conductance and exercise endur-
ance implicates VEGF as a candidate for doping regulations.

Evidence for a Systemic Effect


Despite concern throughout the past decade over whether
PRP might enhance athletic performance, scientific evidence
to support or refute this hypothesis remains limited. At the
time of writing, four studies have been published on the sys-
temic effects of locally administered PRP in humans.
In 2006, Banfi et al. [6] conducted the first investigation
into whether local intratendinous PRP injection was associ-
ated with changes in systemic growth factor concentrations.
Blood was collected in five male patients before, and 30 min,
3 h and 24 h after receiving a single intratendinous leukocyte-
rich PRP injection. They found no significant change in inter-
leukin 4, interleukin 6, interleukin 10, interleukin 1α,
interleukin 1β, tumour necrosis factor α, or interferon γ.
However, endothelial growth factor (EGF) fell significantly
from 30 min to 3 h, and returned to baseline by 24 h.
Chemokine (C-C motif) ligand 2 (CCL2) also fell signifi-
cantly at 3 h. Anitua et al. have also previously reported a fall
in EGF after addition of PRP to human tenocytes in vitro [1].
Although the Banfi study was limited by its short duration,
small sample size, and limited selection of growth factors
studied, it was critical in providing the first evidence to sug-
gest that PRP may have systemic effects beyond the local
tendon environment.
In 2011 and 2012, Schippinger et al. published the results
of an experiment in which 10 healthy male volunteers
received a 2.5 mL intramuscular injection of autologous con-
ditioned plasma [78, 79]. Blood samples were collected
before, and at 30 min, 3 h and 24 h after PRP, and ELISA
assays were performed for IGF-I, EGFs, PDGF, FGFs, VEGF,
and TGFs. The mean circulating levels of IGF-I, PDGF-AB,
PDGF-BB, TGF-β1, VEGF, EGF, and FGF were not signifi-
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 209

cantly different at 30 min or 24 h, relative to the group’s mean


at baseline. However, they did report a statistically significant
threefold rise in circulating TGF-β2 throughout the 24 h after
intramuscular PRP injection. This was the first published
investigation of many of the growth factors explicitly banned
by WADA, including IGF-I, PDGFs, FGF and TGFs.
However, since this study was powered to detect only large
increases in IGF-I (40 %), smaller but potentially meaningful
changes in IGF-I or other growth factors may have missed
the threshold for statistical significance. An additional limita-
tion is that statistical analyses in this study were conducted by
comparing the mean growth factor level across all partici-
pants at baseline to the group’s mean level after PRP. Since
levels were pooled into a single mean value at each time
point, this method fails to account for large variations in
growth factor levels between patients and may miss changes
in growth factors on the individual level.
Recently, Wasterlain et al. measured 6 ergogenic growth
factors monitored by WADA – hGH, IGF-I, IGFBP-3,
bFGF, VEGF, and PDGF-BB – in 25 patients before and at
15 min, and 3, 24, 48, 72, and 96 h after intratendinous
leukocyte-rich PRP injection [90]. Serum levels increased
significantly by 7–8 % for IGF-I at 24 and 48 h, by 1.6- to
2.3-fold for bFGF at 72 and 96 h, and by 30–50 % for VEGF
at 3, 24, 48, 72, and 96 h after PRP injection relative to each
individual’s own baseline. An indirect marker for hGH dop-
ing, the product of IGFBP-3 × IGF-I, also increased signifi-
cantly by up to 35 % after PRP, supporting a possible
ergogenic effect of PRP. Finally, elevated VEGF was observed
in all 25 patients after PRP, and >88 % of patients had ele-
vated VEGF at each time point from 3 to 96 h after PRP,
suggesting that VEGF may be a sensitive molecular marker
to detect athletes recently treated with PRP. Strengths of the
study include: sufficient power to detect a 10 % change in
growth factors, rigorous standards to ensure blood sample
quality, and the comparison of each individual’s post-PRP
growth levels to his or her own baseline to control for inter-
individual variability. This study suffers from some of the
210 A.S. Wasterlain et al.

same limitations as the other 3 studies, namely the lack of an


untreated control group, the relatively short study duration,
the use of a single commercially available PRP (in this case,
Biomet GPS III).
The authors offer two hypotheses to explain their observa-
tion that circulating levels of some growth factors increased
significantly after PRP injection [68]. Since the PRP itself
contained less IGF-I than serum at baseline, the increase in
circulating IGF-I at 24–96 h cannot be from the PRP and
must be from an endogenous source. One possibility is that
the observed increase in circulating IGF-I after local PRP
injection could be due to activation of the hGH-IGF-I axis.
This is supported in part by the significant rise in circulating
IGF-I within 24–48 h, which is consistent with previous stud-
ies showing that IGF-I responds to hGH within the same
time window [9, 31, 51]. Furthermore, 75 % of circulating
IGF-I is secreted by the liver as part of the hGH-IGF-I axis,
and IGF-I is the most specific marker of supraphysiological
GH exposure [20].
Alternatively, one or more of the growth factors contained
within PRP might directly stimulate local production of addi-
tional growth factors. For example, since tenocytes express
the hGH receptor [29] and hGH has direct effects on most
tissues including stimulating IGF-I synthesis [19, 31], it is pos-
sible that the small amount of hGH contained within PRP
binds to tenocyte hGH receptors and stimulates IGF-I syn-
thesis, ultimately contributing to a systemic rise in IGF-I.
Evidence regarding the ability of PRP to stimulate growth
factor synthesis in tenocytes is conflicting. Although one
animal study showed that intratendinous PRP injection
enhances IGF-I gene expression and protein synthesis in the
epitenon and endotenon [58], Anitua et al. [1] found that PRP
induced VEGF but not IGF-I production by human teno-
cytes in vitro. In a transgenic mouse model, Yakar et al. [100]
showed that local tissue IGF-I accounts for only 25 % of the
total circulating IGF-I pool. Therefore, it seems less likely
that local IGF-I production alone could explain the 7–9 %
rise in circulating IGF-I.
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 211

Since none of the aforementioned studies directly


addressed the molecular mechanisms underlying systemic
growth factor changes, the mechanism by which PRP might
activate the hGH-IGF-I axis is unclear. The technique of PRP
injection involves penetrating the tendon, which may cause
local bleeding from the peritenon and could thereby provide
access to the bloodstream. In vitro and animal studies have
demonstrated that PRP triggers an inflammatory response [3,
23], suggesting that PRP contents may be able to signal and
recruit inflammatory cells from distant locations in vivo.
Furthermore, both IGF-I and hGH receptors are ubiqui-
tously expressed, including within capillary endothelium and
in brain parenchyma [73], and IGF-I crosses the blood-brain
barrier [69, 73]. Although these pathways are not completely
understood, these and other studies support possible mecha-
nisms for molecules contained within PRP to participate in
hGH–IGF-I axis stimulation. Additional research is war-
ranted to clarify the biological mechanisms by which PRP
might affect circulating growth factor levels. To assist in
directly establishing an ergogenic effect of PRP, future clini-
cal studies should compare patients treated with PRP to
those in an untreated control group, evaluate the effect of
injection volume, and track athletic performance along with
systemic growth factor changes.

PRP Treatment
Multiple groups have reported that PRP contains elevated
concentrations of growth factors such as VEGF, PDGF, and
FGF [10, 23, 25, 90]. Even though PRP is not itself banned, its
systemic effects could potentially influence results of anti-
doping tests [86, 90]. For example, because PRP causes some,
but not all, of the changes expected after hGH doping, PRP-
treated athletes could mistakenly test positive for
hGH. Another concern about PRP is that athletes could use
PRP as a vehicle to inject exogenous substances, including
hGH. Testing for PRP could deter the intentional use of PRP
212 A.S. Wasterlain et al.

to enhance performance. Together, these issues indicate that


a unique test for PRP may be warranted.
Traditionally, WADA and other antidoping organizations
have relied on the direct detection of markers and metabo-
lites, which are compared with population-based threshold
values. However, the emergence of biological therapies, par-
ticularly endogenous ones such as PRP, and the increasing
sophistication of athlete doping methods have led to a para-
digm shift in antidoping strategies. In 2009, WADA intro-
duced the Athlete Biological Passport, which refers to a
collection of laboratory data points for an individual athlete
that is saved and monitored over time, constituting an indi-
vidual and longitudinal profile. Rather than comparing ath-
lete values to a standardized threshold, each participant
becomes his or her own reference, and deviations from the
athlete’s own norm are flagged. In the future, PRP treatment
could potentially be detected and monitored as part of the
Athlete Biological Passport.

Other Medical Concerns About PRP


Beyond the doping-related issues discussed above, some sci-
entists have expressed concern about other potential medical
issues associated with PRP use. These concerns are generally
theoretical, based on the mechanisms of growth factors con-
tained within PRP, and have not yet been directly assessed in
clinical or laboratory studies. Because PRP may trigger a
systemic rise in TGF-β after intramuscular injection [79],
some researchers have suggested that PRP injection could
induce a fibrotic healing response in local muscle tissue,
which may prevent optimal healing and subsequently lead to
an increased incidence of re-injury [12, 81]. Concerns have
also been raised over the oncogenic potential of IGF-I, which
is not elevated in PRP itself, but increased significantly by
7–8 % in serum following local PRP injection [90]. The asso-
ciation between serum IGF-I levels and prostate cancer has
been demonstrated repeatedly [32, 33], and two large case-
Chapter 9. The Systemic Effects of Platelet-Rich Plasma 213

control studies have revealed a 7–8 % elevation in serum


IGF-I among men prostate cancer relative to age-matched
controls [11, 92]. Similarly, elevated serum IGF-I levels are
associated with a 2.5 relative risk for colorectal cancer [59],
and a dose-dependent risk for lung cancer (2.1 odds ratio)
[102]. Some authors have argued that, since most PRP sys-
tems do not concentrate IGF-I, concerns about its carcino-
genic potential are unwarranted [78]. Notably, the relationship
between cancer and PRP itself has not been directly studied.

Conclusion
Sports physicians are in a unique position to uphold the prin-
ciples of clean competition, as well as to investigate new
technologies, such as PRP, with the potential to facilitate
athletes’ recovery from injury in a manner consistent with
anti-doping regulations. Creaney et al. and the broader sports
medicine community have recognized an ironic dichotomy in
which the general public may benefit from PRP, but recovery
from orthopaedic injuries is delayed in elite athletes because
of doping restrictions [17]. As recognized by the International
Olympic Committee (IOC) in a recent Medical Commission
Consensus Statement on the use of growth factor technolo-
gies in therapy, “Knowledge of the genomic and proteomic
impacts of growth factor-based therapies on the target cells,
and of the biomarkers reflecting stem cell differentiation
status, will underpin the development of tests capable of
monitoring therapeutic efficacy and minimizing adverse
events” [55].
Additional research into the potential systemic and ergo-
genic effects of PRP is warranted to ensure athlete safety, and
so that clear, evidence-based guidelines can be implemented
regarding the use of PRP. Future studies should directly com-
pare circulating growth factors in PRP-treated individuals to
those in untreated control participants. Other potential areas
for research include comparing intratendinous versus intra-
muscular injections; evaluating the effects of dry-needling
214 A.S. Wasterlain et al.

alone to dry-needling with PRP injection over a range of


volumes to address whether a dose-dependent effect may
exist; and tracking systemic growth factor concentrations as
well as athletic performance after local PRP injection to
directly establish an ergogenic effect of PRP.

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Chapter 10
Potential Links Between
Tendon Pathology and
Platelet Rich Plasma Biology
Isabel Andia, Eva Rubio-Azpeitia, and Nicola Maffulli

Introduction

Tendons are a crucial part of the musculoskeletal system. By


connecting muscles to the bones they allow musculoskeletal
movement. But these structures are vulnerable to lesions, espe-
cially in the presence of risk factors, including overuse, ageing,
trauma, metabolic disorders or obesity [1]. Some tendons are
particularly susceptible to tendinopathy; these include Achilles,
patella, elements of the rotator cuff, forearm extensors, biceps
brachi and tibialis posterior tendons. Disorders of these tendons
are often chronic and are complicated to manage successfully.

I. Andia, PhD () • E. Rubio-Azpeitia, MSc


Regenerative Medicine Laboratory, BioCruces Health Research
Institute, Cruces University Hospital, Barakaldo 48903, Spain
e-mail: iandia2010@hotmail.com
N. Maffulli, MD, MS, PhD, FRCS (Orth)
Department of Musculoskeletal Disorders, Faculty of Medicine,
Surgery and Dentistry, University of Salerno, Salerno, Italy
Centre for Sports and Exercise Medicine, Barts and London School
of Medicine and Dentistry, Queen Mary University of London,
London, UK
e-mail: n.maffulli@qmul.ac.uk

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 223


Practice, DOI 10.1007/978-1-4471-7271-0_10,
© Springer-Verlag London 2016
224 I. Andia et al.

Most common existing therapies are physiotherapy, extra-


corporeal shock wave therapy, and local injections of gluco-
corticoids, NSAIDs, autologous blood, or sclerosant injections.
The inadequacy of these therapies to meet the clinical
demand for tendon conditions calls for new research.
Research in the last decade has advanced knowledge
about tendon biology and pathology, and the field has been
stimulated by novel insights and the development of biologi-
cal interventions for injured tendons. Platelet rich plasma
(PRP) is not novel and goes back several decades [40]. PRPs
were deeply rooted in the haematological field since the 70s
[34]. Intravenous systemic transfusions of PRP were used to
combat platelet deficits. They were also used in the 1980s for
the treatment of leg ulcers [26], and later in maxillofacial
surgery to enhance bone formation around dental implants,
accelerating implant osseointegration and consolidation [31].
In the new millennium, PRP was introduced in orthopae-
dics and sports medicine [41], and used extensively to treat
tendinopathies and joint conditions [42]. However, the field
has evolved little, and what we still have is one product for
many targets [22]. Developing formulations specific to indica-
tions require a deep knowledge, not only of how PRP influ-
ences healing mechanisms but also about the conditions of
the host tissue. PRP therapies could be successful only after
refining formulations tailored to the appropriate stage of the
disease to treat.
In this chapter, we will discuss recent progress on under-
standing tendon biology with a particular focus on the break-
down of tendon homeostasis leading to pathology. Secondly,
we will analyse the current knowledge of PRP biology in
relation with the management of tendinopathy.
Current PRP interventions stem from two concepts. The
first is that tendon pathology is consequence of a failed heal-
ing response to tissue microdamage. Secondly, in this context,
the signalling cytokines in PRP can change tissue microenvi-
ronment and enhance healing mechanisms. These concepts
are based on biological hallmarks of tendinopathy, including
stem cell exhaustion and aberrant differentiation, tenocyte
Chapter 10. Tendon Pathology and PRP Biology 225

dysfunction, and altered intercellular communication with


disorganization of extracellular matrix (ECM), and subse-
quent transition from health to disease [3, 7].

Non-healing Mechanisms Involved in Tendon


Pathology

Given the scarce cell number and reduced motility and pro-
liferation rates of tenocytes, and complicated by the absence
of blood vessels, tendons have poor ability to self-repair.
Healing may be also hindered by repetitive mechanical stress
without sufficient time to heal.

Unresolved Inflammation

Growing evidence supports the contribution of inflammation


to the development of tendinopathy [12, 39]. The phenotypes
of key cells orchestrating inflammation in tendinopathy and
enthesopathy have not been fully characterized, but immune
cell players include myeloid cells, i.e. monocyte/macrophages.
Tendinopathic tendons display CD14+ and CD68+ myeloid
cells [13]. Furthermore, early and advanced tendon disease
showed different patterns in the expression of inflammatory
molecules [20].
Initial neutrophil infiltration arises probably as a conse-
quence of the synthesis of IL-8 by local cells, and facilitates
recruitment of monocytes/macrophages. Monocyte infiltra-
tion is also stimulated by the local synthesis of MCP-1 and
RANTES in response to IL-1beta present in the tendon
milieu [4].
Macrophages, conventionally categorized into M1 and M2,
exist in a spectrum of states, depending on their environment,
and play an important role in determining whether the tissue
is in proinflammatory state (M1 activation) or through M2
polarization shifts to a resolution of inflammation.
226 I. Andia et al.

Subclinical inflammation in tendinopathy can be trig-


gered by cell death induced by microdamage. Intracellular
molecules released to the extracellular space upon cell
death originate alarm and tissue stress. Molecules named
Danger Associated Molecular Patterns, DAMPs, embody
the latter. Details about the role of DAMPs (also known as
alarmins) such as high mobility box group 1, heat shock
and S100 proteins, hyaluronic fragments, uric acid, nucleic
acids and mitochondrial components, in tendinopathy are
emerging [33, 37].
DAMPS trigger innate immune cell migration and activa-
tion. Monocytes/macrophages have membrane receptors,
namely TLRs, which bind DAMPS. This binding is the pre-
liminary step to activate various intracellular pathways
including NF-kB signaling, and ensuing the synthesis and
release of inflammatory cytokines such as IL-1beta [28].
Subsequent deregulation of key proinflammatory molecules
(IL-1b, TNF-a) may prolong the inflammatory phase, and
delay healing. In addition, this mechanism has repercussion
for ECM homeostasis (another hallmark of non-healing tis-
sues) because NF-kB-activated cells can be involved in
MMP-mediated ECM damage [8].
Inflammation and remodeling occur rapidly after tendon
overload [43]. Moreover, although tendons lack lymphatic
vessels in physiological conditions, dense lymphatic supply
was present in a rat lesion model [44].

Abnormal Angiogenesis

Angiofibroblastic features observed in histologic images of


tendinopathy are consistent with the hypothesis of incom-
plete resolution of angiogenesis [44]. Indeed, normal heal-
ing is characterized by an angiogenic stage in which blood
vessels proliferate to cope with the nutrient demand of
proliferating cells. This is followed by downregulation in cell
number and regression of the blood vessels [45]. The latter
Chapter 10. Tendon Pathology and PRP Biology 227

is fine-tuned by pro- and anti-angiogenic factors that either


promote or inhibit angiogenesis. Failure of these mechanisms
is a feature of tendinopathy.
Angiogenesis-related cytokines have been proposed as
sensitive markers for early tendon degeneration. The current
paradigm proclaiming that, unlike other tissues, tendon can-
not follow the classical sequence of tissue healing mecha-
nisms because of the lack of innervation and vascularization
is eclipsed by the secretory paracrine capability of local teno-
cytes, which synthesize soluble mediators involved in the
migration of several populations of cells, including innate
immune cells and cells involved in angiogenesis [5, 6].

Alterations in Paracrine Communication

Tissue healing phases can be characterized by coordinated


cell signaling involving different cell systems. Integration of
specific messages derived from distant cells (i.e. neuron) with
messages from cells located, perhaps centimeters apart (the
paracrine system), coupled with signals released from identi-
cal cells, perhaps millimeters away (the autocrine system)
conform an inter-cell communication system the disturbances
of which affect tissue homeostasis. This means that there will
be a variety of cytokines and growth factors that coordinate
a diversity of cell functions in health and disease.
Reception and interpretation of chemical signals will con-
trol cell decisions and their fate. Cells sense specific signals
located in the pericellular space provided that they express
specific transmembrane receptors. Interaction of the signal
with its receptor triggers an intracellular signaling cascade
that reaches the nucleus, and activates gene expression A cell
can be defined by the receptors it uses and these receptors
might vary with adaptation and maturation of the cells. Thus
cells interpret extracellular signals that control tissue fate.
This is the molecular basis of the maintenance or disruption
of tendon homeostasis, and the basis for PRP actions [11].
228 I. Andia et al.

Cell signaling is complex, and one signal may be necessary


but not sufficient: the cell may only react, provided that other
requirements are met. Signaling factors may have inhibitory
or stimulatory actions on cell division, growth and differen-
tiation. Within tissues and organs, molecular signals may be
present within the extracellular matrix. Their effects depend
on the cellular and molecular context. The dynamic spatial
and temporal nature of these mechanisms represents a chal-
lenge when trying to identify critical mechanisms involved in
tendon pathology.

Dysregulation of the ECM Compartment

The ECM theory for the initiation of tendinopathy advocates


that repetitive strains below failure levels produce micro-
damage [47]. Initiation of tendinopathy may therefore be
asymptomatic, but reach a threshold when damage is irrepa-
rable [2].
In most instances, this damage can have marked conse-
quences, as tendons have low healing resources. ECM dam-
age involves the removal of fragmented collagen fibrils and
their subsequent replacement. A tendon can be unable to
maintain its structural integrity under certain conditions and
imbalances can develop between damage generation and its
removal [29]. The subsequent accumulation of damage, failed
healing efforts, and fibrotic scarring shape tendinopathy.

Decrease in the Regenerative Capacity


of Stem Cells
Another plausible hypothesis, alternative to the ECM theory,
is that cellular changes set in motion and lead the way for
matrix alterations [47]. This cell-based theory has been
extended to progenitor cells. It stems from findings showing
that the precursor tendon cells responsible for tendon
homeostasis display hindered or altered differentiation pro-
cesses. Actually, tendon homeostasis is preserved by resident
Chapter 10. Tendon Pathology and PRP Biology 229

tendon progenitor cells, which have several functions [15].


Firstly, they differentiate into tenocytes, and substitute dam-
aged cells. Secondly, they build the damaged ECM.
Overall, this process involves the ability of progenitor cells
to detect alterations in the macromolecular composition and
organisation of ECM, i.e. degraded macromolecules and their
ability to respond by building new matrix.
Mechanical loading modulates all of these cell functions.
For instance, in response to shear stress, tenocyte up-regulate
the production of ECM molecules, while apoptosis, prolifera-
tion and signaling-related genes are largely down-regulated.
These changes involve an anabolic tendon response [35].
However, tenocytes also exhibit a number of changes in
response to loading that are consistent with reduced matrix
production and enhanced matrix degradation. These include
increased apoptosis, and synthesis of inflammatory media-
tors, which have been linked to decreased matrix production,
and increased degradation by MMPs and ADAMTS [21, 25].
It is probable that dysregulated tissue turnover, as a conse-
quence of altered cell activities, is involved in the develop-
ment of tendinopathy [38].
Knowledge of the basic biological mechanisms involved in
tendon response to injury is critical in therapeutic manage-
ment. As healing mechanisms are capable of restoring tissue
homeostasis, deviations from healing mechanisms can be
responsible for pathology. Disordered or insufficient healing
has been hypothesized to lead to tendinopathy. The complex-
ity of failed healing may be challenged with interventions
that target multiple biological processes such as platelet-rich
plasma.

PRPs Deliver Molecular Signals Interfering


with Tendinopathy

PRPs provide a critical opportunity to influence the course of


tendinopathy. As described, below PRP can modulate inflam-
mation and angiogenesis, activate progenitor cell chemotaxis
230 I. Andia et al.

and proliferation. In addition, anabolic molecules in PRP can


enhance the synthesis of collagens and other ECM building
proteins. Based on the PRP definition as MeSH term (intro-
duced in Pubmed in 2007), the effectors of the beneficial
function of PRP therapies are growth factors, such as PDGF,
TGF, FGF, endothelial growth factor (EGF), hepatocyte
growth factor (HGF), connective tissue growth factor, and
VEGF, among others. However, recent proteomic analyses
have identified more than 300 proteins in the alpha-granules
[32]. In addition, the crucial role of specific plasma proteins
in inflammation, such as alpha2macroglobulin, is being
unraveled. Thus, we have to consider reinterpretation of PRP
therapies and analyze the role of molecules other than
growth factors.
In tendon conditions, PRP molecules target not only teno-
cytes located within the rigid fibrillar ECM, but also tendon
fibroblasts, which abound in the endontenon. These cells can
be more prone to migrate and fulfill repair activities, because
a loose ECM surrounds them. Currently, we lack specific cell
markers for the apparently different tendons cells.
As illustrative examples of PRP molecules other than
growth factors, but involved in crucial biological mechanisms
modulated by PRPs, we quote molecules seldom mentioned
in studies of PRPs, such as the chemokine (C-X-C motif)
ligand (CXCL)-7 and platelet factor 4 (PF-4 or CXCL-4) in
innate immune response [23], thrombospondin-1 (TSP-1) in
angiogenesis, and urokinase plasminogen activator (uPA) in
cell migration [10].

Inflammation

Inflammation is an umbrella term that encompasses the func-


tional consequences of innate immune responses.
Inflammation can originate from microtendon injuries, and
the innate immune system mediates the process. Platelets
modulate inflammation largely because of their ability to
secrete high levels of chemokines (a subset of small, diffusible
Chapter 10. Tendon Pathology and PRP Biology 231

cytokines), required to control extravasation of innate


immune cells and their infiltration in the injured tissue.
Supporting this hypothesis, the inhibition of platelet activa-
tion with antiplatelet glycoprotein Ib decreased polymorpho-
nuclear leukocyte influx by 50 %. Platelets are considered the
major source of beta-thromboglobulin (CXCL-7 or
neutrophil-activating peptide-2 [NAP-2]), chemotactic and a
strong activator for neutrophils. In fact, platelets release two
CXCL-7 precursors (i.e. platelet basic protein) and connec-
tive tissue-activating peptide III, but proteolytic processing
by neutrophils is essential to make chemotactically active
CXCL-7. Hence, leukocyte-PRP fibrin is likely to attract
more neutrophils from the bloodstream than PRP fibrin
alone. This issue, although needing experimental confirma-
tion, may be clinically relevant when deciding to use pure
PRPs or leukocyte-platelet concentrates. A priori, we exclude
neutrophils because they may exacerbate tissue damage via
several different mechanisms (such as secreting proinflam-
matory cytokines, such as tumor necrosis factor, interferon
gamma [IFN-g], and interleukins [IL-6 or IL-1b]) that cause
matrix destruction through the production of metalloprote-
ase-1 (MMP-1), -3, and -13. In addition, the interaction of
neutrophils with platelets may induce a hyperactive migratory
response of circulating neutrophils toward the injury site.
Thus, a large influx of these cells to the site of injury and sub-
sequent activation of oxidative and enzymatic processes can
intensify host tissue damage.
Another crucial event in inflammation is removal of
necrotic cells, which depends on the monocyte/macrophage
lineage. Platelets are the major source of PF 4, which prevents
monocyte apoptosis and promotes macrophage differentiation.
PF-4, often ignored when describing PRP composition, is
the most abundant protein in the alpha granules. Recent
experiments using microarray technology have shown that
PF-4 elicits a unique macrophage state of polarization that
shares diverse molecular similarities with both the pro- and
antiinflammatory activation patterns [24]. In tendinopathy,
the biological manipulation of macrophage polarization with
232 I. Andia et al.

PRP may shift the inflammatory state to an anti-inflammatory


healing milieu. This can be a critical topic to advance basic
tendon science and refine PRP therapies.
PRP may terminate inflammation by polarizing macro-
phages to the noninflammatory phenotype; but not only
macrophages, and also local cells, have plasticity and can be
restored to noninflammatory phenotype.
PRPs may terminate inflammation by restoring cells to a
noninflammatory phenotype. This effect could be mediated
by various GFs, including HGF, VEGF, and TGF-b. For
example, HGF treatment induces an antiinflammatory cyto-
kine profile in endothelial cells specifically by suppressing
E-selectin. In a model of inflammatory activation (LPS stim-
ulation of macrophages), the presence of HGF induced a
decrease in the pro-inflammatory cytokine IL-6 and an
increase in the antiinflammatory cytokine IL-10, in part by
interfering with NF-kB signaling [14, 18]. HGF is primarily
found in plasma, and very little is found in platelets. Therefore,
it is important to consider that the therapeutic effects of PRP
result from synergies between platelets and plasma proteins.

Angiogenesis

The role of platelets in dysregulated angiogenesis that occurs


in tendinopathy is intriguing. Platelets contain both stimula-
tors and inhibitors of angiogenesis, thus, when injected in
enthesopathy, characterized by upregulated angiogenesis,
modulation of angiogenesis is the expected therapeutic ben-
efit. However, clinical data reporting modifications of the
angiogenic status of tendon are scarce. In our experience [30],
as assessed by ultrasound, PRP does not modify significantly
angiogenesis in the enthesopathy commonly found in epicon-
dylitis, nor does it exacerbate the angiogenic status.
Platelet alpha granules contain a variety of angiogenic
proteins including VEGF [36], which represents a major rate-
limiting protein during the angiogenic process by binding to
VEGFR-2 in endothelial cells. The concentration of VEGF
depends on the cellular composition of PRPs, as it correlates
Chapter 10. Tendon Pathology and PRP Biology 233

positively, not only with the number of platelets but also with
leukocyte concentration. However, the angiogenic effect of
PRP is not only attributed to pro-angiogenic factors such as
VEGF, and PRP contains molecular stimuli that up-regulate
the production of further angiogenic molecules [9].
Alpha-granules, the main molecular reservoir in platelets,
also contain established inhibitors of angiogenesis, such as
TSP-1, an adhesive protein modulating vascular cell behavior
by altering endothelial and vascular smooth muscle cell adhe-
sion, proliferation, motility, and survival. TSP-1 concentration
is proportional to the number of platelets [48]. TSP-1 inter-
feres with VEGF and bFGF binding, in doing so suppressing
their mitogenic effects. Other antiangiogenic proteins in
PRPs are angiostatin, endostatin, and fibronectin and the tis-
sue inhibitors of metalloproteinases (TIMPs-1 to -4) [46].
Pro- and anti-angiogenic proteins may well be stored sepa-
rately and differentially released because the secretion of
pro- versus anti-angiogenic stores may be agonist-specific.
For example, PAR1-activating peptide, ADP, and the glyco-
protein VI-targeting collagen-related peptide induced mas-
sive release of VEGF but a modest release of PF-4 or
endostatin. In contrast, PAR4-AP triggered marked PF-4 and
endostatin release. This suggests that depending on the
molecular milieu distinct secretion patterns of pro- and anti-
angiogenic factors will be achieved [17]. On this basis, PRP
therapies have the potential to produce selective pro- or
antiangiogenic environment depending on the host tissue
conditions.
Also, often neglected is the fact that a pool of small mol-
ecules, such as histamine, noradrenaline, dopamine, and sero-
tonin (stored in dense granules) can increase vascular
permeability, thereby permitting the extravasation of plasma
proteins into the injury.

Cell Migration and Proliferation

Platelets contain chemokines such as SDF-1a (CXCL12), a


ligand for CXCR4, involved in directed cell migration.
234 I. Andia et al.

However, the potent chemotactic effect of PRP is dependent


on synergic actions between several molecules, as SDF-1a per
se is not responsible of the potent chemotactic properties of
PRPs.
Regulation of the pericellular environment is necessary
for cell migration and matrix remodeling. Platelets contribute
to this aspect by releasing fibrinolytic factors (and their
inhibitors). Among several proteases and protease inhibitors
released by platelets, most notably uPA and plasminogen
activator inhibitor type 1 adjust migratory events and influ-
ence the overall pericellular proteolytic balance [16, 27].
Clearly, the binding of plasminogen activator inhibitor type 1
with its several targets, including vitronectin, uPA, and uPA/
uPAR, has the potential to affect the motile program on mul-
tiple levels, providing the opportunity to therapeutically
manipulate this pathway in pathophysiological settings [19].

Anabolism/Catabolism and Matrix Remodeling

PRP-released anabolic cytokines include connective tissue


growth factor (CTGF), TGF-b1 and -b2, and IGF I among
others. These cytokines have different effects that are tissue-
context dependent. For example, TGF-b1 is a pleiotropic
protein that may influence cell differentiation. In other con-
ditions, TGF-b enhances collagen deposition and has been
associated with fibrotic healing. Remarkably, molecular
changes that occur with aging could lead to growth factor
dysfunction. Actually, the functionality of both TGF-b1 and
IGF-I declines as a result of the aging-related changes in
growth factor–receptor interactions. Assuming impaired ana-
bolic growth factor stimulation with increased age, PRP
actions would differ when administered to older patients. If
this holds true, allogeneic PRP obtained from young adults
could be more effective for old patients.
The biological paradox is that platelets may be, however,
also implicated in matrix destruction for remodelling pur-
poses. Indeed, MMPs are also secreted by platelets, but in an
inactive form, and their activity is regulated by the molecular
Chapter 10. Tendon Pathology and PRP Biology 235

microenvironment. Classically, MMPs are collectively viewed


as capable of degrading all components of the extracellular
matrix and basement membrane, restricting their functions to
tissue remodeling and maintenance. However, extracellular
matrix degradation releases noncovalently bound growth fac-
tors and cytokines, and thereby increases their bioavailability.
Examples include release of VEGF and TGF-b1. VEGF
binds non-covalently to heparan sulfate proteoglycans with
release on extracellular matrix proteolysis. TGF-b is main-
tained in a latent state by binding to the latency-associated
peptide (LAP). LAP, in turn, is covalently bound to the fibril-
lin protein latent TGF-b-binding protein. Extracellular matrix
degradation releases the latent complexes, and dissociation of
the TGF-b–LAP complexes increases TGF-b availability.
Moreover, LAP is a substrate of MMP-2, -9, -13, and -14, and
latent TGF-b-binding protein can be cleaved by MMP-7.
The multitude of potential cellular mechanisms acting
simultaneously in tissue repair or failure helps to explain why
the field can move toward targeting specific pathways by
harnessing the effects of particular molecular subsets present
in PRP preparations. Moreover, PRP biotechnology can be
used for much more than enhancing tissue healing. In the cor-
rect hands, PRPs can also be essential tools for discovering
the decisive molecules in specific healing pathways.

Perspectives

There are no efficacious treatments for tendon conditions


other than palliative. Although the clinical efficacy of PRP in
tendinopathy is controversial, PRP is broadly used. Biological
therapies are at the forefront because they may offer hope in
this unmet medical condition. However, while some studies
testing PRP efficacy showed improved clinical outcomes,
other studies failed to demonstrate relevant outcomes [1, 3, 7].
The lack of knowledge to discriminate subsets within tendi-
nopathic patients or tendinopathy stages hinders progress
and the development of efficacious treatments.
236 I. Andia et al.

Advances in the field are restricted by the heterogeneity


of tendons, involving not only biological and mechanical dif-
ferences between intrasynovial and extrasynovial tendons
but also demographic variability between tendinopathies, i.e.
patellar tendinopathy versus rotator cuff conditions. In this
context, developing knowledge through experimental
research will help to elucidate different biological aspects of
tendinopathy and establish a link between the beneficial bio-
logical properties of PRP.
Progress in translational research would help to establish
at which stage of the condition PRP treatment could be more
effective, or improve our ability to sort patients into pheno-
types. Although common thinking claims that high quality
RCTs are needed to attain definitive conclusions, we accept
as true that limitations to attain critical knowledge hinder the
refinement and design of appropriate clinical trials to test
biological treatments.
These limitations would be subdued if we arrive at a com-
prehensive description of the relation between PRP essen-
tials, pathological components of tendinopathy and healing
mechanisms.

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Index

A clinical application, 116–117


Abnormal angiogenesis, 226–227 defects of, 108
Acetaminophen, 132 dense granules, 110
Achilles tendinopathy, 95 hip application, 114–115
Achilles tendons, 40 knee application, 111–114
Activation procedures, for PRP lisosomal granules, 110
calcium chloride and physiopathology, 107–108
thrombin activation, 17 sources of variability, 109–110
fibrinogen cleavage, 16 in talar osteochondral lesions,
freeze/thaw cycle, 17–18 115–116
in-vivo collagen activation, 18
mechanisms, 16
platelet degranulation, 16 B
Alarmins. See Danger associated Back pain, 49
molecular patterns Basic fibroblast growth factor
(DAMP) (bFGF), 201–202
Alpha-granules, 110, 147–148, Beta thromboglobulin
232–233 (beta-TG), 181
American orthopedic foot and bFGF. See Basic fibroblast
ankle society growth factor (bFGF)
(AOFAS), 98–99 Biological therapy
Anabolism/catabolism, in chemokines, cytokines and
tendons, 234–235 growth factors, 180–182
Angiogenesis, in tendons, clinical applications
232–233 acute ligamentous njuries,
AOFAS. See American 185
orthopedic foot and arthroscopic rotator cuff
ankle society (AOFAS) repair, 186
Articular cartilage healing, muscle injuries, 185–186
162, 164 osteoarthritis and articular
Articular cartilage lesions, 152 cartilage degradation,
alpha granules, 110 186

N. Maffulli (ed.), Platelet Rich Plasma in Musculoskeletal 241


Practice, DOI 10.1007/978-1-4471-7271-0,
© Springer-Verlag London 2016
242 Index

Biological therapy (cont.) Dense granules, 110, 180


tendinopathies, 185 Doping concerns, 200
economical issues, 188–189
future aspects, 189
leucocytes, 180 E
limitations, 187–188 Emcyte PRP system, 66, 67
mechanisms and growth Extra-articular pain generators,
factor release, 183 133–134
platelets, 179–180 Extracellular matrix (ECM)
PRP preparation, 176–178 compartment dysregulation,
wound healing mechanisms, 229
183–184 degradation, 235
BMC. See Bone marrow
concentrate (BMC)
therapy F
Bone healing, of foot and ankle, FGF-2 See Basic fibroblast
153–154 growth factor (bFGF)
Bone marrow concentrate Fibrin, 6–7
(BMC) therapy, 84–85 Fibroblasts, 20–21
Foot and ankle surgery
alpha-granules, 147–148
C PRP effect
Calcium chloride activation, 17 articular cartilage healing,
Cartilage, 46–49, 76–78 162, 164
Cell migration and proliferation, bone healing, 153–154
in tendons, 233–234 chronic Achilles
Cell populations, 179–180 tendinopathy, 159
Cell therapies, 51–52 human subcortical
Cellular environment progenitor cells, 153
collagen, 21 human tendon cells, 155
fibroblasts, 20–21 osseous and soft tissue
systemic effects, 21 healing, 155
tenocytes, 21 plantar fasciitis, 159, 162
Chemokines, 180–181 in rat Achilles tendon,
Chondroitin, 50–51 155–156
Chronic Achilles tendinopathy, risk factors for impaired
159 osseous healing,
Collagen, 21 157–158
Corticosteroids, 132 soft tissue healing, 158,
160–161
surgery, 156
D syndesmotic fusion with
DAMP. See Danger associated agility TAR, 157
molecular patterns soft tissue and cartilage,
(DAMP) 152–153
Danger associated molecular sport injury
patterns (DAMP), 226 angle sprains, 149
Index 243

forefoot, 151 Human tendon cells, 155


hindfoot, 150–151 Hyaline cartilage defects, 152
lateral and medial ankle Hyaluronic acid, 132
stabilities, 150
midfoot, 151
syndesmotic complex, 150 I
tendons, 151–152 IGF binding protein type 3
Freeze/thaw cycle activation, (IGFBP-3), 206
17–18 IGFBP-3. See IGF binding
protein type 3
(IGFBP-3)
G IGF-I. See Insulin-like growth
Glucosamine, 50–51 factor I (IGF-I)
Granules, platelet-derived GF, Inflammation, in tendons,
110 230–232
Growth factors (GF), 200 Injection technique method,
basic fibroblast growth factor, 135–138
201–202 Insulin-like growth factor I
and cytokines, 8–9 (IGF-I), 204
hepatocyte growth factor, Intervertebral disc, 74–76
202–203 Intervertebral disk degeneration
human growth hormone, 83, (IDD), 49–50
203–204 Intra-articular human growth
insulin-like growth factor I, 204 hormone, 83
mechano growth factor, In-vivo collagen activation, 18
205–206
molecular signal delivery by
PRPs, 230 K
platelet-derived growth factor Kellgren-Lawrence classification,
type BB, 207 knee joint OA, 130–131
release timing, 183 Knee osteoarthritis, 99–100, 123
vascular endothelial growth behavioral/psychosocial
factor, 207–208 factors, 139
clinical application of PRP,
111–114
H frequency of injections,
Hepatocyte growth factor 137–138
(HGF), 202–203 injection technique, 135–136
HGF. See Hepatocyte growth long-term follow-up studies, 138
factor (HGF) nociception in, 126–127
hGH. See Human growth pathophysiology of, 124–126
hormone (hGH) patient preparation method,
Hip applications, 114–115 135
Human growth hormone (hGH), patient selection
83, 203–204 exercise programs, 132
Human subcortical progenitor extra-articular pain
cells, 153 generators, 133–134
244 Index

Knee osteoarthritis (cont.) M


healing and regenerative Macrophages, 35
properties, 131 Matrix remodeling, in tendons,
Kellgren-Lawrence 234–235
classification, 130–131 Mechano growth factor (MGF),
pharmacotherapy, 132 205–206
standing radiographs, MGF. See Mechano growth
130–131 factor (MGF)
transcutaneous electrical Molecular biology
nerve stimulation, 132 chemokines, cytokines and
post procedure care, 136–137 growth factors,
research and evidence, 180–182
127–128 leucocytes, 180
short-term follow-up mechanisms and growth
studies, 138 factor release timing,
183
platelets, 179–180
L PRP preparation, 176–178
Lateral epicondylitis (LE), 96 wound healing mechanisms,
Leukocyte-and platelet-rich 183–184
fibrin (L-PRF), 34 Molecular signal delivery by
Leukocyte-and platelet-rich PRPs, 229
plasma (L-PRP), 34 anabolism/catabolism and
Leukocyte-poor PRP (LP-PRP), 12 matrix remodeling,
Leukocyte-rich PRP (LR-PRP), 234–235
11–12 angiogenesis, 232–233
Leukocytes cell migration and
acute inflammatory proliferation, 233–234
response, 4–5 growth factors, 230
antimicrobial role in PRP, 6 inflammation, 230–232
biological therapy, 180 Muscle injury, 44–46
LP-PRP, 12 Muscle pathology, 73–74
LR-PRP, 6, 11–12 Musculoskeletal system
neutrophil granules, 6 applications
rich vs. poor PRP, 65–66 cartilage, 76–78
Ligament pathology, 72–73 intervertebral disc and
Lisosomal granules, 110 spine pathology, 74–76
LP-PRP. See Leukocyte-poor ligament pathology, 72–73
PRP (LP-PRP) muscle pathology, 73–74
L-PRF. See Leukocyte-and tendinopathy, 69–72
platelet-rich fibrin classification system, 64
(L-PRF) preparation
L-PRP. See Leukocyte-and anti-coagulation, 68–69
platelet-rich plasma leukocyte rich vs. poor
(L-PRP) PRP, 65–66
LR-PRP. See Leukocyte-rich platelet activation, 68–69
PRP (LR-PRP) with/without RBCs, 66–68
Index 245

rehabilitation after PRP categories, 34


injections, 78–81 neutrophils and
revised PLRA, 64–65 macrophages, 35
tips and concerns platelets augment anabolic
bone marrow concentrate, signaling, 35
84–85 principles, 34
intra-articular human types of leukocytes, 33
growth hormone, 83 spinal column
pioneer patients, 84 cell therapies, 51–52
ultrasound guided knee discogenic pain, 49
aspiration, of glucosamine and
suprapatellar bursa, chondroitin, 50–51
81–82 intervertebral disk
degeneration, 49–50
tendons and ligaments
N ACL reconstruction
Neutrophils, 35 surgery, 43
Nociception, in knee OA, 126–127 cartilage, 46–49
Non-healing mechanisms, in muscle, 44–46
tendons platelet gel and leukocytes,
abnormal angiogenesis, 44
226–227 sparse vascularization, 42
ECM compartment visual analogue scale, 43
dysregulation, 229 ultrasound-guided anesthetic
paracrine communication, nerve blocks
alterations in, 227–228 Achilles tendon, 38, 40
stem cells, regenerative intra-articular knee
capacity of, 228–229 injections, 38–39
unresolved inflammation, knee, 38–39
225–226 supraspinatus tendon, 38, 40
systemic medication and
physical methods, 38
O ultrasound-guided peripheral
Orthopedics, 41–42 nerve blocks, 36–37
Osseous healing, 157–158 Paracrine communication, in
Osteoarthritis (OA) in knee. See tendons, 227–228
Knee osteoarthritis Patellar tendinopathy, 95–96
Osteochondral defects, articular Patient preparation method, 135
surface lesion, 108 Patient-rated tennis
elbow evaluation
(PRTEE), 96
P Patient selection, in knee OA
Pain medicine exercise programs, 132
orthopedics, 41–42 extra-articular pain
processing generators, 133–134
bovine thrombin, collagen healing and regenerative
and calcium activation, 36 properties, 131
246 Index

Patient selection, in knee OA muscle pathology, 73–74


(cont.) tendinopathy, 69–72
Kellgren-Lawrence in articular cartilage (see
classification, 130–131 Articular cartilage
pharmacotherapy, 132 lesions)
standing radiographs, 130–131 basic fibroblast growth factor,
transcutaneous electrical 201–202
nerve stimulation, 132 for biological therapy
PDGF-BB. Platelet-derived cell populations, 179–180
growth factor type BB chemokines, cytokines and
(PDGF-BB) growth factors, 180–182
Pharmacotherapy, for knee clinical applications,
osteoarthritis, 132 184–187
Plantar fasciitis, 159, 162 economical issues, 188–189
Plantar fasciopathy, 97–99 future aspects, 189
Platelet activation, 68–69 limitations, 187–188
Platelet and leukocyte gel mechanisms and growth
(PLG), 34 factor release time, 183
Platelet-derived growth factor type PRP preparation, 176–178
BB (PDGF-BB), 207 wound healing
Platelet factor 4 (PF4), 181 mechanisms, 183–184
Platelet-poor plasma (PPP), 12–15 classification systems, 19–20, 64
gel, 16 commercial systems, 19
Platelet-rich formulations, 9 content preparations
leukocyte-poor PRP, 12 fibrin, 6–7
leukocyte-rich PRP, 11–12 growth factors and
platelet-poor plasma, 12–15 cytokines, 8–9
PPP gel, 16 leukocytes, 4–6
PRP platelets, 2–4
definitions, 10 doping concerns, 200
gel, 13, 16 effect on cellular environment
Platelet-rich plasma (PRP) collagen, 21
activation procedures fibroblasts, 20–21
calcium chloride and systemic effects, 21
thrombin, 17 tenocytes, 21
fibrinogen cleavage, 16 evidence for systemic effects,
freeze/thaw cycle, 17–18 208–211
in-vivo collagen, 18 foot and ankle surgery
mechanisms, 16 alpha-granules, 147–148
platelet degranulation, 16 articular cartilage healing,
anti-coagulation, 68–69 162, 164
applications bone healing, 153–154
cartilage, 76–78 in chronic Achilles
intervertebral disc and tendinopathy, 159
spine pathology, 74–76 human subcortical
ligament pathology, 72–73 progenitor cells, 153
Index 247

human tendon cells, 155 cartilage, 46–49


osseous and soft tissue definition, 32
healing, 155 muscle, 44–46
plantar fasciitis, 159, 162 orthopedics, 41–42
in rat Achilles tendon, processing, 33–36
155–156 spinal column, 49–52
risk factors for impaired tendons and ligaments,
osseous healing, 42–44
157–158 ultrasound-guided
soft tissue and cartilage, anesthetic blocks,
152–153 37–40
soft tissue healing, 158, ultrasound-guided
160–161 peripheral nerve
sport injury, 149–152 blocks, 36–37
surgery, 156 platelet-derived growth factor
syndesmotic fusion with type BB, 207
agility TAR, 157 preparation
formulations, 9 anti-coagulation, 68–69
definitions, 10 leukocyte rich vs. poor
gel, 13, 16 PRP, 65–66
leukocyte-poor PRP, 12 platelet activation, 68–69
leukocyte-rich PRP, 11–12 with/without RBCs, 66–68
platelet-poor plasma, rehabilitation after PRP
12–15 injections, 78–81
PPP gel, 16 revised PLRA, 64–65
growth factors, 147–148 saftety and use, 116–117
hepatocyte growth factor, stages, 1
202–203 in tendons
human growth hormone, Achilles tendinopathy, 95
203–204 conservative measures, 94
IGF binding protein type 3, knowledge development
206 through research, 236
insulin-like growth factor I, 204 lateral epicondylitis, 96
in knee osteoarthritis, 99–100 molecular signal delivery
clinical applications, by PRPs, 229–235
127–134 non-healing mechanisms,
methods, 134–138 225–229
nociception in, 126–127 patellar tendinopathy, 95–96
pathophysiology of, plantar fasciopathy, 97–99
124–126 rotator cuff, 97
patient selection, 130–134 treatment analysis, 235–236
mechano growth factor, tips and concerns
205–206 bone marrow concentrate,
medical concerns, 212–213 84–85
in pain medicine intra-articular human
benefits, 32–33 growth hormone, 83
248 Index

Platelet-rich plasma (PRP) R


(cont.) RANTES, 181
pioneer patients, 84 Rat Achilles tendon, 155–156
ultrasound guided knee RBCs. See Red blood cells
aspiration, of (RBCs)
suprapatellar bursa, Red blood cells (RBCs), 66–68
81–82 Rehabilitation after PRP
treatment, 211–212 injection, 78–81
vascular endothelial growth Rotator cuff tendinopathy,
factor, 207–208 97, 98
WADA’s prohibition,
201, 202
Platelets S
cell population trapped in SDF1alpha, 181
PRP, 179–180 Soft tissue and cartilage,
cell types, 3–5 152–153
concentration, 4 Soft tissue healing, 155, 158,
counts, 3–4 160–161
cytokines, 2 Spinal column
granule types, 110 cell therapies, 51–52
linear regression analysis, 3 discogenic pain, 49
pro-and anti-angiogenic glucosamine and chondroitin,
factors, 182 50–51
roles of growth factors and intervertebral disk
molecules in PRP, 8 degeneration, 49–50
Post procedure care method, Spine pathology, 74–76
136–137 Sport injury
Post PRP rehabilitation, 79–80 angle sprains, 149
PPP. See Platelet-poor plasma biomechanics, 149
(PPP) forefoot, 151
P-PRF. See Pure platelet-rich hindfoot, 150–151
fibrin (P-PRF) lateral ankle stability, 150
P-PRP. See Pure platelet- medial ankle stability, 150
enriched plasma syndesmotic complex, 150
(P-PRP) tendons, 151–152
Proplatelets, 179–180 transverse tarsal joint, 151
PRP. See Platelet-rich plasma Stem cells, regenerative
(PRP) capacity of, 228–229
PRTEE. See Patient-rated tennis Subtalar joint, in foot and ankle,
elbow evaluation 150–151
(PRTEE) Superficial defects, articular
Pure platelet-enriched plasma surface lesion, 108
(P-PRP), 34 Supraspinatus tendon, 40
Pure platelet-rich fibrin Syndesmotic complex, 150
(P-PRF), 34 Systemic effects
Index 249

cellular environment, 21 regenerative stem cell


doping concerns, 200 capacity, 228–229
evidence for PRP, 208–211 unresolved inflammation,
growth factors, 200–208 225–226
medical concerns, 212–213 patellar tendinopathy,
treatment, 211–212 95–96
phases of healing, 78–79
plantar fasciopathy, 97–99
T PRP effect in foot and ankle
Talar osteochondral lesions, joint, 151–152
115–116 rotator cuff tendinopathy,
Tendinopathy, 69–72 97, 98
Tendons treatment analysis, 235–236
Achilles tendinopathy, 95 Tendon stem cells (TSC), 156
conservative measures, 94 Tenocytes, PRP effect on, 21
knowledge development TENS. See Transcutaneous
through research, 236 electrical nerve
lateral epicondylitis, 96 stimulation (TENS)
and ligaments Thrombin activation, 17
ACL reconstruction Transcutaneous electrical nerve
surgery, 43 stimulation (TENS),
cartilage, 46–49 132
muscle, 44–46 TSP-1, 233
platelet gel and
leukocytes, 44
sparse vascularization, 42 U
visual analogue scale, 43 Ultrasound-guided anesthetic
molecular signal delivery by blocks, 37–40
PRPs, 229 Ultrasound guided knee
anabolism/catabolism and aspiration, of
matrix remodeling, suprapatellar bursa,
234–235 81–82
angiogenesis, 232–233 Ultrasound-guided peripheral
cell migration and nerve blocks, 36–37
proliferation, 233–234 Unresolved inflammation,
growth factors, 230 225–226
inflammation, 230–232
non-healing mechanisms
abnormal angiogenesis, V
226–227 VAS. See Visual analog scale
ECM compartment (VAS)
dysregulation, 229 Vascular endothelial growth
paracrine communication, factor (VEGF),
alterations in, 227–228 207–208
250 Index

VEGF. See Vascular endothelial World Anti-Doping Agency


growth factor (VEGF) (WADA), 200–202
Visual analog scale (VAS), 43, 96 Wound healing mechanisms,
183–184

W
WADA. See World Anti-Doping
Agency (WADA)