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Platelet-Rich Plasma Preparations for

Biological Therapy: Applications and LimitsGiuliana Gobbi, PhD, and Marco Vitale, MD

Platelets are anucleate blood cells characterized as primary effectors of hemostasis. The

rationale for the therapeutic use of platelets as a surgical adjuvant is to make platelet-derived

factors locally available for tissue healing. Several platelet-derived growth factors have been

recently characterized, able to favor both wound healing and angiogenesis. Biological therapies

using platelet-rich plasma (PRP) preparations are currently being used, making it essential to

expand our knowledge on the sequential events that characterize PRP action. Studies on the

efficacy of PRP in human subjects are still scarce, probably because of the relatively recent

clinical applications of PRP. In some case control studies and in several noncontrolled clinical

trials, PRP has been found effective. However, the results of most studies are hampered byrelevant confounding variables such as the variations of PRP characteristics even in patients

with similar platelet counts. PRP essentially acts as a growth factor reservoir, inducing mito-

genesis, chemotaxis, and angiogenesis at the site of application. However, notwithstanding

several different characteristics between them, all platelet-enriched products are called PRP,

which makes the distinctions difficult. Hence, although PRPs represent a promising tool of

clinical application, many questions are still open, such as the appropriate indications for its

clinical use as well as the effective concentrations and quantities for each product to be used

in each therapeutic situation.

Oper Tech Orthop 22:10-15 2012 Published by Elsevier Inc.

KEYWORDS platelets, PRP, growth factors, tissue regeneration

Platelets: Origin and Release

Platelets derive from megakaryocytes (MKs), giant cellsresident in the bone marrow. Platelets release in sinusoidvessels is subsequent to MKs cytoplasm reorganization infiliform extensions, called proplatelets.1 Granules synthe-sized and assembled in the MK cell body are transported tothe proplatelets (Fig. 1) and stored in the nascent plateletsbefore their release into the circulation.2 At the final stage,platelets are anucleate cells functionally characterized as pri-mary effectors of hemostasis3 containing many granules ofdifferent types (Fig. 2).

Platelet granules contain storage pools of active sub-stances, which are released after adhesion to extracellular

matrix components, such as collagen, or in response to sol-

uble agonists.

There are 3 main types of platelet granules:

1. Dense granules: they are released by exocytosis and

contain several active substances, such as ADP/ATP,

serotonin, and Ca2. P2Y1 and P2Y12 receptors bind

the released ADP, which stimulates platelet activation

and hemostasis4; serotonin mediates vasoconstriction

and capillary permeability,5 whereas calcium is essen-

tial for fibrin formation.6

2. Alpha granules: platelet-secreted proteins, such as

growth factors, chemokines, and cytokines, are stored

in the alpha-granules.7 Released chemokines enhance

recruitment of hematopoietic cells, such as monocytes

and neutrophils, to the vascular wall, participating in

vessel repair and regeneration after vascular injury,8-10

whereas secreted cytokines, such as interleukin-1, -6,

and -8, induce inflammatory response in endothelial

cells.11,12

3. Lysosomes: platelet lysosomes contain enzymes able to

induce protein and matrix degradation.13,14

Human Anatomy Section, Department of Anatomy, Pharmacology and Fo-

rensic Medicine, University of Parma, Parma, Italy.

Supported by MIUR grant FIRB RBAP10KCNS_002 to M.V.

Present address of Giuliana Gobbi: Department of Medicine, Harvard

Medical School, Brigham & Womens Hospital, Boston, MA 02115.

Address reprint requests to Marco Vitale, MD, Human Anatomy Section, De-

partment of Anatomy, Pharmacology and Forensic Medicine, University of

Parma, via Gramsci 14 43126Parma, Italy. E-mail: marco.vitale@unipr.it

10 1048-6666/12/$-see front matter 2012 Published by Elsevier Inc.doi:10.1053/j.oto.2012.01.002

mailto:marco.vitale@unipr.itmailto:marco.vitale@unipr.it

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Platelets asCarriers of Growth Factors

Platelets release many bioactive substances responsible forattracting macrophages, mesenchymal stem cells, and osteo-blasts, not only facilitating the removal of necrotic tissues butalso participating in the biological functions of tissue repairand regeneration. This implies a complex orchestration ofcell differentiation and angiogenesis, ultimately leading towound healing.15-17 The alpha granules released in the heal-ing process by the activation of platelets play a key role inthese processes. Proteomic analysis of the platelet secretome

showed that more than 300 proteins can be released, onlysome of which are well characterized18 and summarized inTable 1. Although both cell differentiation and angiogenesisare essential processes in tissue repair and regeneration, theyare both implicated in cancer development as well.

Platelet-derived growth factor (PDGF)-A and -B (the 2 pre-dominant forms of PDGF in platelets), vascular endothelial

growth factor (VEGF), insulin-like growth factor (IGF)-1,fibroblast growth factor, hepatocyte growth factor, and epi-dermal growth factor have a proangiogenic activity, and theirsecretion can be promoted by CD40L/CD40 interaction.19

CD40L, in turn, promotes endothelial cell proliferation andmigration.20At the site of vessel injury, the release of plateletfactors increases vessel permeability and leukocyte rolling onendothelial cells21 and promotes blood and vascular cell mi-gration and proliferation as well as spouting of new vessels.22

However, other factors secreted by platelets can have theopposite effects on angiogenesis, as is the case for transform-ing growth factor (TGF)-123 and thrombospondin-1,24

whereas platelet factor 4 interferes with the binding of VEGFand other growth factors to cells.8 Other antiangiogenic pro-teins stored and released from platelets include angiostatin,endostatin, and tissue inhibitor of metalloproteinase-1and -4.3,25,26 Pro- and antiangiogenic factors are stored in dif-ferent subsets of alpha granules,27 whose secretion is differentlyregulated by selective engagement of the thrombin receptorsproteinase-activated receptor (PAR)-1 and PAR-4.28 Plateletgrowth factors that favor osteoclastogenesis, such as interleu-

kin-1 and macrophage inflammatory protein-1, are also lo-calized in alpha granules.29-31 IGF-1 stimulates bone matrix

Figure 1 Preformed granules are transported from the megakaryo-

cyte body on microtubule tracks within the proplatelets.

Figure 2 The granules are captured by the developing platelets be-fore their release into the circulation.

Table 1 Main Secretory Proteins Contained in Platelet Alpha

Granules Involved in hemostasis, Chemotaxis, Angiogene-

sis, and Osteogenesis

Fibrinogen (Fg) Hemostasis

Vitronectin (Vn) Hemostasis

Fibronectin (Fn) Hemostasis

Platelet factor 4 (PF4) Antiangiogenic activity

Angiostatin Antiangiogenic activityEndostatin Antiangiogenic activity

Tissue inhibitor of

metalloproteinases (TIMP)-1

and -4

Antiangiogenic activity

Thrombospondin (TSP)-1 Antiangiogenic activity

Transforming growth factor

(TGF)-

Antiangiogenic activity

Chemotaxis

Platelet-derived growth factor

(PDGF)-AA, -BB, and -AB

Proangiogenic activity

Chemotaxis

Fibroblast growth factor (FGF) Proangiogenic activity

Chemotaxis

Hepatocyte growth factor (HGF) Proangiogenic activity

ChemotaxisVascular endothelial growth

factor (VEGF)

Proangiogenic activity

Chemotaxis

Epidermal growth factor (EGF) Proangiogenic activity

Chemotaxis

Insulin-like growth factor (IGF)-1 Proangiogenic activity

Chemotaxis

Osteogenesis

Bone morphogenetic protein

(BMP)-2, -4, and -6

Osteogenesis

Chemotaxis

Macrophage inflammatory

protein (MIP)-1

Osteoclastogenesis

Chemotaxis

Interleukin (IL)-1 Osteoclastogenesis

Osteocalcin (Oc) Osteogenesis

Osteonectin (On) Osteogenesis

Platelet-rich plasma preparations for biological therapy 11

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formation and proliferation of osteoblasts and their precur-sors.32 MKs synthesize bone morphogenetic proteins-2, -4,and -6, which are released by platelets and are essential forbone formation.33

Platelet-Rich PlasmaProduction and Characterization

Platelet-rich plasma (PRP) preparations were originally de-veloped in blood transfusion units to separate red blood cellsand plasma from whole blood. PRP was first used for hemo-stasis during surgical operations,34 and it has also been re-ferred to as platelet-rich concentrate, autologous platelet gel,and platelet releasate.35 These products have been used totreat wounds since 1985.36

PRP contains a high number of platelets and a full array ofclotting and growth factors,35 and, therefore, has mitogenic,chemotactic, and angiogenic properties.37,38 Platelets are har-

vested and concentrated from whole blood and transformedinto an easy-to-handle gel to allow topical application. Sev-eral methods used to prepare PRP and gel are aimed to obtaina high yield of harvested platelets. Given a baseline plateletconcentration of 200,000 platelets/l, PRP is defined as thevolume of the plasma fraction from autologous blood havinga platelet concentration more than baseline.35,37 Clinical effi-cacy of PRP can be expected at platelet concentrations 4 to 6times higher than baseline (1 million platelets/l).38,39

Active PRP is prepared immediately before use by a simple2-step procedure. Step 1 consists of 2 centrifugations, thefirst designed to eliminate red blood cells and the second toenrich plasma with platelets (minimum 1 106/l to obtainclinical efficacy). Step 2 involves activation of the plateletswith the addition of thrombin or calcium chloride, resultingin fibrin polymerization and production of a gelatinous plate-let gel, which is applied to the surgical site with a syringe.

The several products available in the market to obtain au-tologous PRP can differ from each other in the preparationprocedure and results. Different systems, in fact, have differ-ent yields in terms of concentrated viable platelets, with thesedifferences accounting for many of the criticisms regardingthe efficacy of PRP.40 However, most, if not all, of these prod-ucts are collectively called PRP, which does not allow distinc-tion between the different systems and protocols. All avail-

able PRP preparation techniques have some points incommon: blood is collected with anticoagulant usually dur-ing surgery and is processed in about 1 hour.

Several characteristics should be considered to classifyplatelet concentrates.41 The user-friendly ergonomy of prep-aration kits and instrumentation used (the centrifuge). Theseare significant factors when considering the repetitive use ofthese techniques in daily surgical practice. The nature of thefibrin network of PRP is mainly dependent on concentrationof the fibrinogen present in the preparation.42 Low-densityfibrin gels are easy to apply but do not offer sufficient stability tothe support the gel matrix. On the contrary, high-density

fibrin gels have the mechanical stability necessary to displaytheir therapeutic potential for a several-day period. To this

respect, the biomechanical properties of the fibrin networkare of the utmost importance. A rapid polymerization of fi-brinogen produced by high thrombin concentrations leads toa rigid network of monofibers which do not represent theideal matrix to trap cytokines and allow cell migration; on thecontrary, a slow fibrin polymerization will produce a flexiblematrix that will guarantee such functional characteristics on

which the therapeutic applicability and efficacy of PRP arebased. Similarly to extracellular matrix, fibrin first absorbsthen releases platelet-derived growth factors with a kineticthat depends on fibrin structure and has an impact on growthfactors availability to tissues. In turn, fibrin structure de-pends on the pro-coagulant enzyme used to induce the gelformation, together with individual fibrinogen concentra-tion.43 Finally, the leukocyte content of PRP can vary, butthere are not sufficient data available to demonstrate func-tional differences between leukocyte-rich and leukocyte-poor PRP, even though some local antibacterial effects can beassumed in the former instance.

The presence of leukocytes in PRP is a matter of debate.According to some authors, leukocytes should be discardedfrom the preparation to prevent inflammatory processes.44

However, most of the PRPs used contain leukocytes,45 andthere is no good scientific reason to discard them.46

PRP as a DeliverySystem of Growth Factors

The rationale for the therapeutic use of platelets is to makeplatelet-derived factors locally available for tissue to behealed.47-49 As described earlier in the text, in the past fewyears, several systems for platelet gel preparation have be-come commercially available, and, despite the increasingtherapeutic use of PRP, its reported clinical effects are quitevariable.50,51

Platelets present in PRP function as a tissue sealant, initi-ating wound repair,52 whereas fibrin matrix acts as drug de-livery system slowly releasing platelet-derived bioactive fac-tors.53,54 A variety of growth factors are present in PRP,including VEGF, TGF-1, IGF, and PDGF.55,56 PRP plateletsare initially activated by thrombin and collagen, releasinggrowth factors that attract undifferentiated cells into thenewly formed matrix and trigger cell division.57 PRP can (i)

inhibit cytokine release from macrophages, improving tissuehealing and regeneration by limiting the inflammation58; (ii)promote new capillary growth59; and (iii) accelerate epitheli-alization48 in chronic wounds. PRPs also have a role in thelocal nonspecific immune response, as they produce signal-ing proteins that attract macrophages60 and contain a variableamount of leukocytes,45 which have demonstrated antimi-crobial activity against Escherichia coli, Staphylococcus au-reus,61,62 Candida albicans, and Cryptococcus neoformans.62

For all these reasons, it is important to evaluate the growthfactor release in PRP; however, there is a lack of publishedinformation on this topic. Platelets tend to be immediately

activated and growth factors massively released during thefirst hours after application at the surgical site, which may

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explain the lack of mid-term and long-term significant effectsof PRP.63Another problem is represented by the consistencyof PRP, which, in most cases, tends to dissolve quickly. Thereis a high individual variability in platelet growth factors con-tent, which is not necessarily proportional to the plateletcount.64,65 Hence, platelet count alone cannot be predictiveof the growth factor content in individual PRP preparations.

Moreover, platelets are extremely sensitive to any kind ofstress, from blood drawing to PRP gel production.66-68 Thus,the amount of platelet-derived factors available at the end ofthe manipulation process depends on cumulative effects onplatelets, along the entire preparation process.

Mazzucco et al69 studied the growth factors present inplatelet gels obtained by 3 commercially available devicesand a manual procedure, in relation to the influence of sev-eral phases of the production process. A high variation wasfound in the concentration as well as in the release kinetics ofPDGF-BB, TGF-1, b-fibroblast growth factor, VEGF, epi-dermal growth factor, IGF-1, and bone morphogenetic pro-

tein-2 measured after 20 minutes, 1 hour, and 7 days after gelformation had occurred, in relation to the method used. Evensimilar methods for platelet gel preparation revealed differentperformances concerning growth factor recovery and the ki-netics of its release from the gel. In agreement with Weibrichet al,55 they did not find any correlation between plateletconcentration and growth factor concentration in PRP,whereas others did.52 In theory, several factors might contrib-ute to this lack of correlation, including variable susceptibil-ity of platelets to manipulation-induced stress and microag-gregates in PRP impairing platelet counts. Furthermore,growth factorabsorbing proteins might affect both platelet

count and growth factor measurement.41,70

Finally, the role of leukocytes in platelet concentrates isanother controversial issue.71 Beside the aforementioned mi-crobicidal role, leukocytes produce VEGF,72,73 promotingangiogenesis. Ehrenfest et al74 compared pure platelet-richplasma (P-PRP) and leukocyte- and platelet-rich fibrin (L-PRF). These 2 products differ in terms of fibrin structure,leukocyte content, growth factor release (TGF-1, PDGF-AB,and VEGF), and matrix proteins (fibronectin, vitronectin,and thrombospondin-1). The P-PRP gel membranes com-pletely dissolve in the culture medium after 5 days, whereasthe L-PRF membranes last more than 7 days, during whichthey slowly release significantly larger amounts of all bioac-

tive factors than the P-PRP gel membranes. Overall, the po-lymerization and molecular architecture of the fibrin matrixand the presence of leukocytes considerably influence thestrength and the growth factor trapping/release potential ofthe membrane.

Current Applications of PRP

The various available platelet concentrates will present dif-ferent biological characteristics, although it is unclearwhether these differences are clinically relevant. Only a mi-nority of clinical studies specify platelet concentration in PRP

used to treat patients, and most of them were not controlledtrials.75

Autologous PRP has less safety concerns than cell-basedregenerative therapies, hence the interest it has generated fortissue engineering and regenerative medicine. The therapeu-tic use of PRP was pioneered in dentistry,76 whereas morerecently, its clinical applications expanded to other fields,such as cardiac surgery,77 ophthalmology,78 oral and maxill-ofacial surgery,79 orthopedic surgery,80 plastic surgery,81,82

sports medicine,83-85 and cosmetic medicine.86,87 PRP admin-istration to target lesions has been also applied for arthritis,meniscal injury, and rotator cuff tears,51,83,88 more on thebasis of empiricism and anecdotal reports than solid scientificknowledge.

From a certain point of view, PRP potentiates the naturalhealing process, releasing multiple growth factors in theirbiologically determined ratios. Both the cellular and molec-ular components of PRP are autologous, as opposed to theuse of recombinant human growth factors; from this perspec-tive, recent studies linked the overexpression of PDGF-BB tomalignant transformation in human cells.89 Autologous PRP

is safer than allogenic preparations and gives no concernsover transmissible diseases,35,40,90 such as human immuno-deficiency virus, hepatitis, and so forth, and is not immuno-genic for the host.35

Finally, the use of PRP may be more cost-effective andeconomical than other therapeutic approaches.

Conclusions

Although PRPs represent a formidable tool for clinical appli-cation, a lot of questions are still open, such as the appropri-ate indications for its clinical use as well as the effective con-centrations and quantities for each product to be used in eachtherapeutic situation. Given what has already been demon-strated and what is promised based on the available data interms of benefits for the patients, a special research effort inthe PRP field will be worth its costs.

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