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

� 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 cor-

responds 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 out-

comes 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.

Keywords Platelet-rich plasma � Bone � Bone healing �Growth factors � 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 investi-

gation 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 terminol-

ogy 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 stud-

ies. In addition, current techniques, terminology, and

practical considerations are reviewed to provide a

clearer indication for the use of PRP in bone healing

specifically.

A. Malhotra � M. H. Pelletier � Y. Yu � 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

123

Arch Orthop Trauma Surg (2013) 133:153–165

DOI 10.1007/s00402-012-1641-1

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 macro-

phage, 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 regener-

ation [11]. Typically, many of these molecules are pro-

duced 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 insta-

bility [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,

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 che-

motactic and mitogenic growth factors, hemostatic factors,

adhesion molecules, and other cytokines [14]. Dense

granules perform a primary role via the release of pro-

aggregating 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 estab-

lished roles in the regulation of tissue regeneration. Platelet

adhesion to endothelial cells is promoted by adhesive glyco-

proteins secreted from a-granules, such as fibronectin, vitro-

nectin, thrombospondin and von Willebrand factor [14, 16],

with fibronectin and vitronectin also promoting osteogenic

cell adhesion and spreading [17]. Although complex syner-

gistic 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 cytoplas-

mic 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 intracel-

lular 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 neces-

sary 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

154 Arch Orthop Trauma Surg (2013) 133:153–165

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(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 con-

nective tissue cells, most notably fibroblasts and chondro-

cytes [23, 24]. Supporting this, the involvement of PDGF

in angiogenesis via the promotion of endothelial cell pro-

liferation [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 increas-

ing 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 associ-

ated receptors expressed on endothelial cells, a cellular

response is induced in which released matrix metallopro-

teinases (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

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 chon-

drocytes 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 com-

peting 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, chon-

droblasts, 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 endo-

chondral bone formation [42], demonstrated by the high

expression in the cartilaginous phase [43, 44]. The osteo-

genic 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 dis-

plays 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

Arch Orthop Trauma Surg (2013) 133:153–165 155

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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 chon-

drocytes 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 heal-

ing. 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 reg-

ulating osteoblast mitosis and differentiation [62].

The stimulation of migration and proliferation of endo-

thelial 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 upreg-

ulating 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, how-

ever, 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.

IGF is sourced from the bone matrix, endothelial cells,

osteoblasts, chondrocytes and platelets, with the presence

of BMPs possibly stimulating the secretion of IGF. Pro-

liferation 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 mor-

phogenetic 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 doseand time dependent. Whencomparing

rhBMP, rhPDGF, and rhVEGF, Kaipel et al. [51] reported

that only rhBMP supported bone regeneration, with both

156 Arch Orthop Trauma Surg (2013) 133:153–165

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rhVEGF and rhPDGF failing to improve healing above the

fibrin matrix control. The use of recombinant growth fac-

tors 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.

Platelet-rich plasma

History

The separation of blood components for surgical applica-

tion 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

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, con-

clusive indication for the use in bone healing still remains

to be established.

PRP production

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 aggrega-

tion more effectively for the use with PRP production

Fig. 1 Evidence for growth factor relationships to the stages within the endochondral healing pathway

Arch Orthop Trauma Surg (2013) 133:153–165 157

123

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 centrifu-

gation 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 col-

lected 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 autolo-

gous blood with platelet count above baseline, this defini-

tion 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 for-

mation 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

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 pro-

cessing; 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 ligand-

binding 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, asso-

ciated 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 colla-

gen, 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 superna-

tant 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

158 Arch Orthop Trauma Surg (2013) 133:153–165

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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 supplemen-

tary 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 postop-

erative 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 che-

motactic 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 degrada-

tion [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 concen-

tration is central in any PRP product. In addition, the leu-

kocyte and fibrinogen concentrations, and activation

methods used, should be routinely reported in PRP prod-

ucts [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.

Practical applications

The basic elements for bone tissue engineering are sig-

naling 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 outper-

formed 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 recog-

nized 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 dis-

traction 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 cal-

varia 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]

Arch Orthop Trauma Surg (2013) 133:153–165 159

123

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 osteo-

inductive 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 addi-

tion 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

the synthetic graft and PRP alone, and even when com-

pared 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 avail-

ability 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 osteogen-

esis 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 com-

bined 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 com-

bination of PRP and rhBMP should also be investigated.

Fig. 2 Platelet secreted growth

factor interactions with major

cell types within the bone

healing timeline

160 Arch Orthop Trauma Surg (2013) 133:153–165

123

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 osteocon-

ductive 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 clo-

sely 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 com-

monly 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 dif-

ferent 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 syn-

ergistic benefit of combining PRP to autograft or allograft.

However, the addition of PRP to synthetic bone graft

substitutes (BGS) appears to be beneficial in some instan-

ces, 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 The authors declare that they have no conflict

of interest.

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