This document reviews the use of platelet-rich plasma (PRP) to improve bone healing. It summarizes the bone healing process and the growth factors involved. PRP contains concentrated platelets that release growth factors important for bone regeneration like PDGF, VEGF, TGF-β, and BMPs. While PRP has potential to enhance healing based on growth factor delivery, studies show inconsistent results. PRP may be most beneficial when combined with osteoconductive scaffolds, but high concentrations and aggressive processing methods do not necessarily improve outcomes. Many variables influence PRP efficacy which should be considered when using it for bone healing.

1. Arch Orthop Trauma Surg (2013) 133:153–165
DOI 10.1007/s00402-012-1641-1
ORTHOPAEDIC SURGERY
Can platelet-rich plasma (PRP) improve bone healing?
A comparison between the theory and experimental outcomes
Angad Malhotra • Matthew H. Pelletier
Yan Yu • William R. Walsh
•
Received: 12 July 2012 / Published online: 30 November 2012
Ó Springer-Verlag Berlin Heidelberg 2012
Abstract The increased concentration of platelets within
platelet-rich plasma (PRP) provides a vehicle to deliver
supra-physiologic concentrations of growth factors to an
injury site, possibly accelerating or otherwise improving
connective tissue regeneration. This potential benefit has
led to the application of PRP in several applications;
however, inconsistent results have limited widespread
adoption in bone healing. This review provides a core
understanding of the bone healing mechanisms, and corresponds this to the factors present in PRP. In addition, the
current state of the art of PRP preparation, the key aspects
that may influence its effectiveness, and treatment outcomes as they relate specifically to bone defect healing are
presented. Although PRP does have a sound scientific
basis, its use for bone healing appears only beneficial when
used in combination with osteoconductive scaffolds;
however, neither allograft nor autograft appear to be
appropriate carriers. Aggressive processing techniques and
very high concentrations of PRP may not improve healing
outcomes. Moreover, many other variables exist in PRP
preparation and use that influence its efficacy; the effect of
these variables should be understood when considering
PRP use. This review includes the essentials of what has
been established, what is currently missing in the literature,
and recommendations for future directions.
A. Malhotra Á M. H. Pelletier Á Y. Yu Á W. R. Walsh (&)
Surgical and Orthopaedic Research Laboratories,
Prince of Wales Clinical School,
The University of New South Wales, Sydney, Australia
e-mail: w.walsh@unsw.edu.au
A. Malhotra
e-mail: angadmalhotra@live.com
M. H. Pelletier
e-mail: m.pelletier@unsw.edu.au
Keywords Platelet-rich plasma Á Bone Á Bone healing Á
Growth factors Á Tissue engineering
Introduction
Healthy bone has the capacity to repair and remodel itself,
however, complications continue to impair functional
repair resulting in delayed healing and non-unions. In an
attempt to reduce the risk of complications, surgical
intervention is frequently undertaken for trauma cases
presenting moderate to massive loss of bone stock, soft
tissue damage, disruption of surrounding vasculature,
and/or infection. The standard surgical technique for bone
repair has previously been achieved via stable fixation in
combination with the gold standard of autogenous bone
grafting [1]; however, the associated risk of donor site
morbidity, increased operative time, blood loss, and length
of hospitalization have encouraged the continual investigation into alternatives [2].
Since the reported success of platelet-rich plasma (PRP)
combined with autograft in treating mandibular defects [3],
PRP has found increasing enthusiasm across a diverse
range of fields. Similarly, it presents yet another ambitious
option for bone healing. Despite the hype, contrasting
surgical outcomes compounded with conflicting terminology and descriptions [4, 5] have limited the widespread
adoption of PRP.
This review presents the basic science of bone healing
and platelets, thus providing a logical basis to discuss
findings previously reported in animal and human studies. In addition, current techniques, terminology, and
practical considerations are reviewed to provide a
clearer indication for the use of PRP in bone healing
specifically.
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Bone healing
Complications relating to bone healing often arise from the
extensive formation of fibrocartilage, resulting in delayed
unions or non-unions affecting approximately 5–10 % of
cases. Failure to heal within 3 months is considered
delayed, although non-union is considered as a failure to
unite within 6–9 months [6, 7]; however, due to the lack of
investigation parameters available other than radiology and
clinical appearance, no consensus exists on the point of
actually diagnosing such healing complications.
Cases requiring surgical intervention typically heal via
the endochondral ossification pathway [8], which is
generally divided into four consecutive, but overlapping
phases: hematoma formation, soft callus formation, hard
callus formation, and remodeling. This process is initiated by
cells of the immune system, whereby a hematoma forms, and
inflammation ensues. Following this, neovascularization and
fibrous tissue formation leads to the development of hyaline
cartilage, forming the soft callus. This soft callus undergoes
cartilage mineralization and subsequent formation of woven
bone which defines the hard callus. Finally, the conversion of
the hard callus to functional lamellar bone progresses via
continuous bone remodeling [9].
The inflammatory phase of bone healing is regulated by
pro-inflammatory cytokines secreted by invading macrophage, polymorphonuclear leukocytes and lymphocytes
[8]. The expression of tumor necrosis factor-a (TNF-a) and
interleukin-1 (IL-1) peaks at 24 h post-injury, activating
secondary signaling pathways involved in the downstream
processes involved in callus formation [10]. Platelets are
activated during this early phase, and in combination with
fibrin, form the hematoma. Upon activation, platelets
secrete a variety of cytokines which have been attributed to
successful hard and soft tissue development and regeneration [11]. Typically, many of these molecules are produced and secreted by cells from a variety of tissues, in
which most circulate within the blood. In the case of bone
healing, platelet activation and subsequent degranulation
provides a burst of cytokines directly at the injury site.
Although delayed bone healing and non-unions may be
exacerbated by a variety of interpersonal factors, including
pre-existing diseases, medication, cigarette smoking, age, and
infection, most commonly these healing complications are
associated with vascularization issues and mechanical instability [12]. The role of specific growth factors relate to these
aspects, being angiogenesis and endochondral bone formation.
Platelets
The platelet life cycle begins with the differentiation of
hematopoietic stems cells on the endosteal bone surface,
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producing megakaryocyte progenitors that migrate to the
blood vessels within the bone marrow. The formation of
proplatelet extensions from megakaryocytes into the vessel,
and subsequent proplatelet maturation results in *2.5 lm
proplatelet fragments being released within the vasculature to
circulate as platelets [13]. Within platelets, three main platelet
secretory granules exist: a-granules; dense granules; and
lysosomes.
The most prevalent a-granules comprise approximately
10 % of the platelet volume, and upon degranulation,
deliver hundreds of proteins either into the extracellular
matrix or expressed as membrane bound proteins on the
surface. These proteins are composed of an array of chemotactic and mitogenic growth factors, hemostatic factors,
adhesion molecules, and other cytokines [14]. Dense
granules perform a primary role via the release of proaggregating factors, such as calcium ions and adenosine
diphosphate (ADP) [15], and lysosomes are involved in the
release of clearing factors in the form of digestive enzymes
[16]. Selectively, these molecules have crucial and established roles in the regulation of tissue regeneration. Platelet
adhesion to endothelial cells is promoted by adhesive glycoproteins secreted from a-granules, such as fibronectin, vitronectin, thrombospondin and von Willebrand factor [14, 16],
with fibronectin and vitronectin also promoting osteogenic
cell adhesion and spreading [17]. Although complex synergistic connections exist between the various molecules [18],
the release of specific growth factors has primarily driven the
prospect of platelets for tissue regeneration.
Growth factors
Growth factors generally perform their function through
the binding of ligands to the associated extracellular
receptors on target cells, leading to intracellular cytoplasmic proteins attaching to the phosphorylated tyrosine.
Although independent intracellular activation pathways
exist [19], ligand binding to the receptor tyrosine kinase is
most commonly associated with the downstream intracellular signaling via growth factors. This process is followed
by a series of phosphorylation and activation steps of
protein kinases within the cytoplasm. The final step
involves the translocation of a phosphorylated kinase to the
cell nucleus, phosphorylating transcription factors necessary for the transcription of genes [20, 21]. Ultimately, this
complex pathway results in the stimulation, or inhibition,
of cell migration, proliferation and differentiation.
Growth factors of particular relevance to this review are
platelet-derived growth factor (PDGF-AB, -BB), vascular
endothelial growth factor (VEGF-A), hepatocyte growth
factor (HGF), the transforming growth factor superfamily,
including transforming growth factor b1, b2 and b3

3. Arch Orthop Trauma Surg (2013) 133:153–165
(TGF-b1, -b2, -b3) and bone morphogenetic proteins (BMP),
fibroblast growth factor (FGF), and insulin-like growth factor
(IGF). Although these are commonly presented as specific
purpose factors, the crosstalk between separate and various
signaling pathways are complex and not as easily defined.
Platelet-derived growth factor
The significance of PDGF is the ability to initiate callus
formation through the chemotaxis of mesenchymal stem
cells [22], and the chemotaxis and mitogenesis of connective tissue cells, most notably fibroblasts and chondrocytes [23, 24]. Supporting this, the involvement of PDGF
in angiogenesis via the promotion of endothelial cell proliferation [25], and the chemotaxis of neutrophil and
macrophage which may provide a secondary stage of
growth factor release, highlights PDGF as a crucial initiator
of bone healing [26].
The three isoforms of PDGF with the most understood
roles in bone healing are constructed with A and B chains:
PDGF-AA; -AB; -BB; with the associated platelet-derived
growth factor receptors (PDGFR) being either a- or
b-subunits. Although different isoform binding affinities
exist, the A chain is able to bind only to a-receptors,
whereas the B chain binds to both a- and b-receptors.
Because higher levels of b-receptors are expressed in
general than a-receptors, the PDGF-AB and PDGF-BB
dimers are considered more potent proteins than the -AA
isoform [26], with specifically PDGF-BB gaining increasing attention for bone healing over other PDGF isoforms
[27]. As a reference, platelets contain PDGF in a ratio of
60–70 % PDGF-AB, 20–40 % PDGF-BB, and 5–25 % of
PDGF-AA [26, 28].
Vascular endothelial growth factor and hepatocyte
growth factor
Angiogenesis is a highly regulated process with brief
periods of action, and then complete inhibition [29]. This
process is essential for successful healing by providing
oxygen and nutrients to the injured site via the newly
formed blood vessels. The significance of VEGF is in its
clear role in neovascularization as a potent endothelial
chemokine and mitogen. Once VEGF binds to the associated receptors expressed on endothelial cells, a cellular
response is induced in which released matrix metalloproteinases (MMP) digest the surrounding extracellular
matrix. This matrix degradation allows for the migration
and proliferation of vascular endothelial cells essential for
the formation of the new blood vessels [30].
Although VEGF target cell receptors are contained
primarily on endothelial cells, the expression of VEGF
receptors by chondrocytes in the epiphyseal growth plate
155
demonstrates the involvement of VEGF in bone formation,
lengthening and endochondral ossification [31, 32]. VEGF
release from platelets has been well established [33–35],
with additional VEGF release from hypertrophic chondrocytes also assisting in the timely angiogenic signaling
necessary for the transition from soft to hard callus [32].
The role of hepatocyte growth factor (HGF) in bone
healing is yet to be elucidated. One possibility is an
involvement in angiogenesis, where VEGF signaling
pathways are activated through the HGF receptor, c-Met,
inducing similar endothelial cell responses without competing with the VEGF surface receptors [36]. HGF may
also be involved as a positive regulator of angiogenesis by
working synergistically with VEGF [36, 37]. Although the
role of HGF in osteogenesis is also uncertain, it has been
shown to be expressed during bone healing, promoting the
osteogenic differentiation of MSC [38], and stimulating
BMP signaling through the upregulation of BMP receptors
on MSC [39]. Based on the above, the attraction of HGF
for bone healing appears to be in the indirect, synergistic
roles promoting angiogenesis and osteogenesis.
Transforming growth factor b
The TGF-b superfamily consists of structurally and
functionally related factors regulating many biological
processes, including cell growth, differentiation, adhesion,
migration and apoptosis. This superfamily has been
strongly associated with many of the bone healing
processes, and comprises TGF-b (1–3), BMPs, growth
differentiation factors (GDF), activins, and inhibins [40].
TGF-b is a polypeptide that stimulates the proliferation
of fibroblast and MSC, with three isoforms being expressed
in humans, TGF-b (1–3). Although platelets constitute a
major source of TGF-b, production by osteoblasts, chondroblasts, and macrophage result in a significant deposit of
TGF-b in bone [41]. The commonly recognized role of
TGF-b is the promotion of chondrogenesis during endochondral bone formation [42], demonstrated by the high
expression in the cartilaginous phase [43, 44]. The osteogenic potential has also been recognized [45], signifying
TGF-b as a stimulator of both chondrogenic and osteogenic
MSC differentiation [46]. These properties, combined with
its involvement in osteoclast apoptosis and inhibition [47],
associates TGF-b with the critical early and mid-stage
processes in the endochondral bone healing pathway.
All three of the isoforms of TGF-b are highly relevant;
collectively, their expression has been reported through
many of the crucial bone healing processes. TGF-b1 displays a constant moderate expression throughout bone
healing, with greater involvement in osteoblast mitosis. In
contrast, TGF-b2 and b3 expression peaks strongly during
chondrogenesis, with the b2 isoform being possibly the
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most potent of the two, exhibiting high expression within
the proliferative, hypertrophic, and mineralization phases
[43, 45, 48].
Since the pioneering work of Urist [49], one of the most
cited proteins within the bone healing field has been the family
of bone morphogenetic proteins (BMP). BMPs are known to
be potent osteoinductive proteins involved in many of the
processes related to bone formation and regeneration [50].
Osteoprogenitors, osteoblasts, mesenchymal cells, and chondrocytes deposit BMPs within the extracellular matrix, where
these growth factors drive MSC differentiation, particularly
down osteogenic lineages [18, 50].
Although evidence supporting the role of BMPs in bone
healing exists [51–53], a therapeutic release of BMPs from
platelets has yet to be established [54]. Platelets were
previously considered to have no true osteoinductive
potential as they were thought not to contain any BMPs
[55], however, BMP-2, -4, -6 and -7 have been found to be
released by platelet concentrates, possibly encouraged by
acidic environments [54, 56]. Despite this finding, the
therapeutic benefit of these endogenous BMPs is unclear;
commercially available exogenous BMP concentrations
used for bone healing applications are commonly quoted at
three or more orders of magnitude greater than those
reportedly released from platelet concentrates [57, 58].
Fibroblast growth factor and insulin-like growth factor
Although the members of FGF family are involved in a
variety of biological functions, the relevance to bone
healing is in the FGF stimulated signaling of MSCs down
osteogenic pathways, and in particular osteoblastogenesis
[46, 59]. FGF may also have an important role during the
remodeling phase of bone healing [60]. Although many
FGFs have been identified with differential temporal
expression within bone healing, two groups of FGF
receptors (FGFR) have particular relevance to bone healing. High expression of both the FGFR1 and FGFR2 on
osteoblasts during hard callus remodeling [61] supports the
assertion that FGF signaling has an important role in regulating osteoblast mitosis and differentiation [62].
The stimulation of migration and proliferation of endothelial cells by FGF-2 [63] suggests that FGFs may also have
beneficial angiogenic properties for bone healing. FGF-2 may
have an indirect, synergistic role in angiogenesis, by upregulating VEGF expression [64]. Asahara et al. [65] reported
such a synergistic effect when combining VEGF and FGF-2
in an ischemic rabbit hind leg model. More recently, however, Willems et al. [66], failed to show any synergistic
angiogenic effect when combining VEGF and FGF-2 with
allograft in a rat segmental bone defect model. As with many
of the growth factors, the required dose of FGF-2 to induce
the intended effect remains unclear.
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IGF is sourced from the bone matrix, endothelial cells,
osteoblasts, chondrocytes and platelets, with the presence
of BMPs possibly stimulating the secretion of IGF. Proliferation and maturation of chondrocytes to hypertrophy is
a pivotal process in the endochondral pathway, and is
regulated by IGF [11, 67]. IGF may also have a role in the
later stages in bone maturation and remodeling [68].
The variety of relevant factors secreted from platelets
forms the basic premise for the use of the product loosely
defined as PRP for bone healing. Although the platelet
lifespan of 8–10 days [13] is considerably less than the
timespan of bone healing, growth factor entrapment within
the fibrin matrix [69, 70] may facilitate the time release of
factors at the healing site; consequently, growth factor
action could outlive the platelet.
Figure 1 illustrates the relationship between platelet
secretory factors to a timeline of the endochondral bone
healing process. In brief, after hematoma formation,
platelet proinflammatory cytokines, such as interleukin-1,
-8, and platelet factor 4 are involved in inflammatory cell
chemotaxis and endothelial–leukocyte adhesion [71].
Chondrogenic differentiation of MSC leads to soft callus
formation characterized by cartilage formation. Cartilage is
calcified before chondrocyte hypertrophy and apoptosis
leads to chondroclast released enzymes facilitating matrix
degradation. Platelet-derived matrix metalloproteinases
(MMP) may also have a role in matrix degradation [72].
Subsequent neoangiogenesis, osteoclast population, and
differentiation of osteoprogenitor cells facilitate the
remodeling of the callus to structural lamellar bone [8, 73–76].
The growth factors presented relate, where shown, to the
stages of endochondral bone healing.
Recombinant growth factors
Although synergistic and antagonistic growth factor actions
are likely to exist, the application of singular exogenous
growth factors provides an accessible and convenient
source of signaling molecules that have the potential to
improve bone healing. Recombinant human bone morphogenetic proteins (rhBMP) have had promising results
for bone healing in both preclinical models [57, 77–79] and
clinical studies [58, 80], however, the efficacy of its use in
all applications remains inconclusive [81, 82]. Although
the use of rhBMP has gained the most interest for bone
healing, rhPDGF also has also had reported success for
similar applications. Recombinant human PDGF-BB
(rhPDGF-BB) is also commercially available, and has been
reported to have a positive effect for bone formation
[83–86]; although, as with many biological therapies, this
effect is likely to be dose and time dependent. When comparing
rhBMP, rhPDGF, and rhVEGF, Kaipel et al. [51] reported
that only rhBMP supported bone regeneration, with both

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157
Fig. 1 Evidence for growth factor relationships to the stages within the endochondral healing pathway
Platelet-rich plasma
finding led to an increased interest and use of PRP within
the oral and maxillofacial surgical fields [89–91]. Since this
early adoption during the 1990s, PRP has seen prolific use
across an increasing variety of surgical fields, to now
include applications ranging from soft tissue healing
[92, 93], cosmetic surgery [94, 95], burns [96], nervous
tissue [97, 98], and chronic skin ulcers [99]. Although the
range of potential applications continues to increase, conclusive indication for the use in bone healing still remains
to be established.
History
PRP production
The separation of blood components for surgical application has a long history; the collection of fibrinogen to use as
intraoperative fibrin glue aiding topical hemostasis found
applications in many clinical settings [87]. Although the
advantages of a hemostatic and adhesive fibrin glue are
known, in 1994, Tayapongsak [88] reported the formation
of the fibrin matrix also supported mandibular bone
remodeling by functioning as a cell supporting scaffold.
Leading on from this, the identification of platelet secreted
growth factors led to the development and use of PRP,
initially reported in 1998 by Marx as beneficial for use in
bone regeneration of mandibular defects [3]. This positive
The production of PRP begins with an autologous blood
sample being needle drawn from a clear venipuncture, and
mixed with an anticoagulant to prevent clotting. Although
a citrate-based anticoagulant may be used, such as sodium
citrate or citrate–phosphate–dextrose [100], Acid–citrate–
dextrose solution A (ACD-A) is most commonly used in
PRP preparations. ACD-A is capable of maintaining the
intraplatelet signal transduction mechanisms during PRP
preparation, and therefore maintaining the responsiveness
of platelets [101]. Ethylenediaminetetraacetic acid (EDTA)
has had reported success in minimizing platelet aggregation more effectively for the use with PRP production
rhVEGF and rhPDGF failing to improve healing above the
fibrin matrix control. The use of recombinant growth factors remains promising, however, as specific temporal
expression of different factors has been observed over the
time course of bone healing [8, 22, 48], the application of
multiple growth factors may more accurately reproduce a
normal healing environment.
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protocols [102], however, EDTA has not been traditionally
recommended for use due to the potential for irreversible
structural, biochemical and functional damage to platelets
[103]. The high level of aggregation inhibition by EDTA
may also restrict future platelet activation, a feature
essential for therapeutic PRP applications.
Although plateletpheresis [104] and filtration [105]
methods do exist, PRP is generally collected after the
separation of the components of whole blood by table-top
centrifugation. The production of PRP by centrifugation
was originally achieved by a two-step gradient centrifugation method. In this method, a hard first spin was used to
separate the red blood cells (RBC) from the plasma which
contained leukocytes, platelets and clotting factor. The
plasma was then centrifuged in a second soft spin intended
to finely separate the platelets and leukocytes [106], after
which PRP was collected. Commonly, and most often with
commercially available systems [107, 108], a one-step
method is employed where the aim is to separate the RBCs,
buffy coat, and plasma into three distinct layers. The buffy
coat contains platelets and leukocytes, and is often collected as a PRP. The plasma layer above is often called the
platelet poor plasma; however, depending on centrifugation
parameters and the collection inefficiency of the technique,
this layer may contain a substantial number of platelets.
The benefit of using commercial systems over manual
methods may be limited to improved ergonomy and
repeatability, rather than platelet collection efficiency.
Regardless of a manual single- or double-spin technique,
the centrifugal forces applied, and length of time at those
forces, presents yet another variable; a variable that highly
influences the platelet concentration. Clinically, any
reduction in time without the loss of quality is obviously
desirable, with the range reported in most studies lies
within 160–3,0009g for 3–20 min [109]. Although PRP
may be defined as a portion of plasma fraction of autologous blood with platelet count above baseline, this definition does not give a full insight into the optimal platelet
count of PRP. Many authors still quote a definition of PRP
by Marx, as a product with platelet concentration of
1,000 9 109/L in 5 mL of plasma [106]. Normal baseline
whole blood platelet count is considered to be around
200 9 109/L, and although studies have reported use of
2–8 times above baseline, a platelet count at 5 times
baseline is often mentioned to be of therapeutic benefit
[55, 110]. Araki et al. [102] compared various manual
single and double-spin preparation methods of PRP,
achieving a maximal 20-fold increase using a double-spin
technique of 2309g for 10 min, followed by pellet formation during a second spin at 2,3009g for 10 min, and
finally pellet resuspension.
Although a high platelet concentration seems to be the
ultimate goal of PRP, the cost of getting there may be
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considerable. Dugrillon et al. [111] reported a decrease in
TGF-b release at forces above 8009g when spun for
15 min, suggesting a possible decline in platelet function at
high G-forces. Poor growth factor release is often attributed
to pre-mature activation and platelet damage during processing; as such, aggressive processing techniques with the
aim of very high platelet concentrations may result in a
paradoxically inferior PRP. Weibrich et al. [112] studied
the effect of platelet concentration on peri-implant bone
regeneration, and concluded that platelet concentrations
between two- and sixfold increase were beneficial, with no
benefit being detected at lower or higher fold increases.
Similarly, Graziani et al. [113] reported a 2.5-fold increase
was optimal for osteoblast proliferation in vitro, however,
all PRP concentrations were still inferior to the positive
control of Dulbecco’s modification of Eagle’s medium
with 10 % fetal calf serum. The potential of growth factors
to transduce a cellular response is limited by the expression
of associated receptors on target cells. Therefore, as ligandbinding sites are finite, excessive platelet concentrations
resulting in excessive growth factor release may not be
beneficial.
The activation method of PRP before implantation has
not been standardized in practice. The simplest option is to
implant the PRP in an anticoagulated state; the theory
behind this approach being that PRP will activate when in
contact with exposed collagen in damaged tissue. In regard
to ex vivo activation, bovine thrombin was previously
considered a suitable PRP activator [114], however, associated complications have all but removed it from use for
this purpose [115]. The use of calcium chloride has also
been reported [116], and may represent a simple, easily
available alternative for clot activation.
Different agonists, such as ADP, thrombin, and collagen, interact with individual platelet surface receptors,
leading to distinct intracellular signaling by messenger
molecules to the separate granules [71]. This results in a
differential granular release depending on agonist. The
thrombin concentration available to the PRP during gel
formation also affects the platelet release, rate of fibrin
formation, the fibrin structure [117], and clot stability
[118]. As such, the production and use of autologous
thrombin is gaining popularity. Autologous thrombin is
produced by collecting the thrombin containing supernatant of a calcium chloride clotted PRP [119], and presents
itself as a useful PRP activator. To date, studies comparing
the effect of different PRP activation methods are lacking.
Leukocytes and fibrin
Leukocyte inclusion and the leukocyte concentration is a
factor often overlooked in many studies. PRP collected
from the buffy coat layer has been reported to contain

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around a sevenfold increase in leukocytes [120]. Castillo
et al. [108] reported 1.7- to 5.6-fold increases in leukocytes
from three commercially available systems.
Growth factors are produced by neutrophils, monocytes
and macrophage, and provide an additional source of
growth factors [23, 121]. Platelets also have a role in the
recruitment of inflammatory cells, such as neutrophils and
monocytes [122]. With regard to immunity, platelets are
themselves known to interact directly with viruses, bacteria
and fungi, and contain platelet microbicidal proteins within
the a-granules [14, 122–124], thus providing supplementary actions to leukocytes.
An increase in leukocytes, combined with the view of
platelets themselves as innate inflammatory cells with
acute host defense functions [122], suggests a PRP product
containing leukocytes may also be useful against postoperative infections. A PRP derivative developed by Anitua
et al. [125] aims to avoid the pro-inflammatory effects of
leukocytes for treating muscle damage. The exclusion of
leukocytes for bone healing, especially in cases of open
injury, is yet to be fully justified.
Fibrin induces angiogenesis by providing a matrix
scaffold which supports cell migration and provides chemotactic activity. The structure of a fibrin clot affects its
ability to perform as a suitable scaffold for cellular
attachment [117], although the binding of thrombin and
growth factors to the fibrin fibers also support healing as a
standby release mechanism during primary clot degradation [69, 70]. The density and composition of the fibrin
matrix is therefore another factor of the PRP [109] not
often considered. Figure 2 illustrates a timeline of bone
healing through the endochondral pathway, highlighting
some of the platelet secreted growth factor interactions
with the relevant cell types within the pathway.
Terminology
Although PRP is a generic term, many terms and acronyms
have appeared to differentiate PRP constituents and state of
activation, but may be also increasing the confusion.
Although many authors urge standardization, the variety of
names unfortunately does little to help standardize the
product. At minimum, four components of PRP should be
reported. Most obviously, reporting the platelet concentration is central in any PRP product. In addition, the leukocyte and fibrinogen concentrations, and activation
methods used, should be routinely reported in PRP products [109, 126]. It is clear that these four variables alone
allow many possible variants of PRP to be produced;
however, provide a simple baseline for comparison. PRP is
used in this review as a blanket term, as previous studies
often do not mention leukocyte concentration, fibrinogen
concentration, and/or activation methods.
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Practical applications
The basic elements for bone tissue engineering are signaling molecules, cells, and matrices [127]. PRP provides
signaling molecules in the form of the variety of growth
factors, and possibly a cell supporting matrix in the form of
the fibrin matrix.
When considering the fibrin matrix, when degradation of
a scaffold does not align with the rate of bone regeneration,
healing may be impaired by either a lack of a scaffold, or
an excessive volume of intact scaffold. When treating
segmental defects placed in the radius of rabbits, Hokugo
et al. [128] reported improved healing when PRP was
combined with a biodegradable gelatin hydrogel, compared
to either PRP alone or gelatin alone. In addition, they
reported that although PRP combined with fibrin outperformed gelatin alone, free PRP was inferior to the gelatin
alone, highlighting the need for PRP to be combined with a
cell supporting matrix. Although the fibrin clot structure
and stability are known factors, the capacity of a PRP gel to
act as the sole scaffold does not appear reliable for bone
healing. To further facilitate cell attachment, the addition
of bone graft substitutes to PRP may be essential for bone
applications. This ensures a suitable scaffold exists during
healing to support cell attachment.
Allogenic and autologous grafts have long been recognized as grafting options. Allogenic demineralized bone
matrix (DBM) is known to often have both osteoinductive
and osteoconductive potential [129], yet, Ranly et al. [130]
reported PRP added to DBM decreased its osteoinductivity.
Similarly, Ni et al. [131] reported the combination of DBM
and PRP was not beneficial over DBM alone during distraction osteogenesis of rabbit tibia. Depending on the
processing, autologous grafts often have osteoinductive,
osteoconductive, and osteogenic properties [132]. These
three properties encompass the prescribed features for
successful tissue healing [127]; hence, the addition of PRP
to autogenous graft may not be beneficial. Mooren et al.
[133, 134] reported no detectable benefit from the addition
of PRP to autogenous grafts in two separate studies in goat
critical size frontal bone defects. Aghaloo et al. [135] also
reported no detectable advantage of combining PRP to
autograft compared to using autograft alone in rabbit calvaria defects.
Conversely, Dallari et al. [136] reported the three part
combination of allograft, bone marrow-derived stem cells
(BMSC), and PRP improved bone regeneration in critical
size distal femur defects in rabbits when compared with
any combination of only two elements alone. In addition,
PRP alone was inferior to either allograft alone or BMSC
alone; however, when PRP was combined with either
allograft or BMSC, was able to improve the healing
response of either component alone. Hakimi et al. [137]
123

8. 160
Arch Orthop Trauma Surg (2013) 133:153–165
Fig. 2 Platelet secreted growth
factor interactions with major
cell types within the bone
healing timeline
also reported a beneficial effect from the combination of
autograft and PRP in tibial metaphysis defects in mini-pigs
when compared with autograft alone. It is not yet clear
whether the addition of PRP to allograft, autologous MSC,
or autograft is beneficial.
Allogenic and autogenic grafts both have some osteoinductive potential. Although PRP is not considered to be
highly osteoinductive in itself, the addition of PRP may be
beneficial to grafts lacking osteoinductivity, as in synthetic
bone graft substitutes (BGS). As synthetic BGS alone have
been shown to support bone healing [138, 139], the addition of biological activity may have the potential to further
facilitate or accelerate healing. Kasten et al. [140] treated
critical sized diaphyseal radius defects in a rabbit model,
and although autologous graft outperformed the test
groups, higher bone formation was reported when PRP was
combined with hydroxyapatite (HA) as compared to the
HA graft alone. Similarly, Kanthan et al. [141] reported
PRP was only beneficial when combined with artificial
osteoconductive scaffolds for the treatment of non-uniting
segmental tibial defects in rabbit. In a clinical case study,
Paderni et al. [142] reported using PRP combined with a
hydroxyapatite-based bone substitute to treat a bifocal
ulnar bone defect. The authors attributed the success of the
graft to the factors present in the PRP, combined with the
osteoconductive hydroxyapatite scaffold. The addition of
bone marrow aspirate to the combination of PRP and
synthetic graft has been reported to improve the rate of
spinal fusion and stiffness in sheep when compared with
123
the synthetic graft and PRP alone, and even when compared with autograft [143]. PRP may have the ability to
introduce osteoinductive potential to a synthetic graft;
however, although platelet–graft interactions may also
exist [144], the optimal synthetic osteoconductive scaffold
to use with PRP remains unclear.
In contrast to autologous PRP, the commercial availability of recombinant growth factors allows for specific
growth factors of known concentrations to be applied to
bone defect sites, thus allowing the ability to consistently
replicate positive outcomes. Recombinant BMP currently
appears the most encouraging for bone healing. As the
theoretical basis of PRP relies on the release of growth
factors other than BMPs, the advantage of PRP application
over rhBMP is uncertain. Hu et al. [145] demonstrated the
potential of both PRP and rhBMP-4 to promote osteogenesis in vitro, however, this effect has not translated to
´
in vivo studies. Roldan et al. [146] compared rhBMP-7 and
PRP, and reported that although the addition of rhBMP-7 to
allograft was able to enhance bone formation, PRP combined with allograft was not. Similarly, Forriol et al. [147]
reported PRP alone was inferior to rhBMP-7 combined
with allograft. Although these studies report the benefit of
recombinant BMP over PRP, the use of PRP combined
with allograft, or PRP alone without an osteoconductive
scaffold, has been shown not be conducive for PRP
effectiveness. Further studies are needed which compare
exogenous growth factor application and PRP. The combination of PRP and rhBMP should also be investigated.

9. Arch Orthop Trauma Surg (2013) 133:153–165
With the current focus on platelet concentration, the
actual volume of PRP to use is often overlooked. Nagata
et al. [116] detected healing differences relating to the ratio
of autograft to PRP volume in critical size defects in rat
calvaria; however, further comparative studies are needed
to ascertain the optimal ratio of PRP volume to graft
volume.
Currently, the use of PRP may be most appropriate for
bone healing when combined with a synthetic osteoconductive scaffold, reducing the need for allogenic products
or autologous harvest of additional tissue or cells. Further
research is required to provide more detailed clinical
indication for use.
Future directions
The establishment of therapeutic doses of platelet
concentration would aid greatly in guiding clinicians in
treatments involving PRP. Ideally these would be in vivo
animal studies that would allow in-depth analysis of bone
regeneration capacity of comparative treatments with closely controlled conditions. PRP platelet concentrations are
difficult to quantify in a clinical situations where coulter
counters or other platelet counting mechanisms are not
readily available. Currently, there are several variables
involved in PRP preparation, making the supposed goal of
1,000 9 109/L difficult to insure, let alone achieve. Purely
focusing on the concentration with disregard for the final
PRP volume may also be distracting, as the volume of the
bone void to be treated will affect the final concentration of
platelets per bone void volume. This effect is not commonly mentioned, and should be considered. Many systems
and studies still report the centrifugal force as revolutions per
minute (RPM), although not reporting the relative centrifugal
force (RCF). It is not possible to compare RPM from one study
to another, as different models of centrifuges will have different rotational radii, making comparisons between methods
and outcomes even more challenging.
It is clear that standardization of terminology and
methods would allow meaningful comparisons between
future studies. The leukocyte concentration and fibrin
structure vary between production and activation methods,
and should be noted. The inherently safe, autologous nature
of PRP has led to its adoption in an ever increasing range of
applications; however, uncertainty in its efficacy does
exist. A greater understanding of the mechanisms and
variables involved may help explain the discrepancies seen
in the translation from preclinical studies to clinical use.
Theoretically, the potential of PRP is great. Despite
completing an intensive and comprehensive literature
research, there is a lack of evidence confirming any synergistic benefit of combining PRP to autograft or allograft.
161
However, the addition of PRP to synthetic bone graft
substitutes (BGS) appears to be beneficial in some instances, and could be recommended if the alternative is the
synthetic BGS alone. The use of PRP alone without any
additional components does not appear to benefit bone
healing, and cannot be recommended. With proper use,
aseptic application of autologous PRP appears to safely
provide access to growth factors that may be useful for
bone healing. Further studies are needed to establish
whether PRP combined with a synthetic BGS has a bone
healing capacity comparable to autograft.
Conflict of interest
of interest.
The authors declare that they have no conflict
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