Arch Orthop Trauma Surg (2013) 133:153–165
DOI 10.1007/s00402-012-1641-1


Can platelet-rich plasma (P...

Bone healing
Complications relating to bone healing often arise from the
extensive formation of fibrocartilage, result...
Arch Orthop Trauma Surg (2013) 133:153–165

(TGF-b1, -b2, -b3) and bone morphogenetic proteins (BMP),
fibroblast growth fac...

most potent of the two, exhibiting high expression within
the proliferative, hypertrophic, and mineralization phases
Arch Orthop Trauma Surg (2013) 133:153–165


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

protocols [102], however, EDTA has not been traditionally
recommended for use due to the potential for irreversible
Arch Orthop Trauma Surg (2013) 133:153–165

around a sevenfold increase in leukocytes [120]. Castillo
et al. [108] reporte...

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

Fig. 2 Platelet secreted growth
factor interactions with major
cell types...
Arch Orthop Trauma Surg (2013) 133:153–165

With the current focus on platelet concentration, the
actual volume of PRP to ...
14. Blair P, Flaumenhaft R (2009) Platelet alpha-granules: basic
biology and clinical correlates. Blood Rev 23(4):177–...
Arch Orthop Trauma Surg (2013) 133:153–165
49. Urist MR (1965) Bone: formation by autoinduction. Science
















Arch Orthop Trauma Surg (2013) 13...
Arch Orthop Trauma Surg (2013) 133:153–165
118. Weisel JW (2007) Structure of fibrin: impact on clot stability.
J Thromb Ha...
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Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes.


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Arch Orthop Trauma Surg. 2013 Feb;133(2):153-65. doi: 10.1007/s00402-012-1641-1. Epub 2012 Nov 30.
Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes.
Malhotra A, Pelletier MH, Yu Y, Walsh WR.
Surgical and Orthopaedic Research Laboratories, Prince of Wales Clinical School, The University of New South Wales, Sydney, Australia.

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Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes.

  1. 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: A. Malhotra e-mail: M. H. Pelletier e-mail: 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. 123
  2. 2. 154 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, 123 Arch Orthop Trauma Surg (2013) 133:153–165 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. 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 123
  4. 4. 156 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. 123 Arch Orthop Trauma Surg (2013) 133:153–165 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
  5. 5. Arch Orthop Trauma Surg (2013) 133:153–165 157 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. 123
  6. 6. 158 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 123 Arch Orthop Trauma Surg (2013) 133:153–165 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
  7. 7. Arch Orthop Trauma Surg (2013) 133:153–165 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. 159 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. 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. 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 References 1. 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