1 CellP R E S S

Classification of platelet concentrates:from pure platelet-rich plasma (P-PRP)to leucocyte- and platelet-rich fibrin(L-PKrJ C<.IL-V^S ~-2oDavid M. Dohan Ehrenfest, Lars Rasmusson and Tomas AlbrektssonDepartment of Biomaterials, institute of Clinical Sciences, The Sahlgrenska Academy at University of Gothenburg, Sweden

*

The topical use of platelet concentrates is recent and itsefficiency remains controversial. Several techniques forplatelet concentrates are available; however, their appli-cations have been confusing because each method leadsto a different product with different btSloyy ai'iU puujriftfat'uses. Here, We present classification of the differentplatelet concentrates into four categories, depending ontheir leucocyte and fibrin content pure platelet-richplasma (P-PRP), such as celt separator PKP, vtvostatPRF—ui—Anilua's PRGf; leUCOcyte- and platelet-richplasma (L-PRP), such as curasan, Kegen, Plateltex,SmartHKeP, PCCS, Magellan or GPS_PRPL-pure piale-tet-ncn ttbrtn (P-PKI-J, such as Fibrinet; and leucocyte-and platelet-rich fibrin (L-PRF), such as Choukroun'sPRF.This classification snouia help to elucidate successesand failures that have occurred so far, as well as provid-ing an objective approach for the further development ofthese techniques.

History and techniquesIn transfusion medicine, platelet concentrates were origin-ally used for the treatment and prevention of haemorrhagedue to severe thrombopenia, which is often caused bymedufiar aplasia, acute leukaemia or significant bloodloss during Jong-lasting surgery. The standard plateletconcentrate for transfusion has been named platelet-richplasma (PRP) and classically contains 0.5« 1G11 plateletsper unit.

The use of blood-derived products to seal wounds andstimulate healing started with the use of fibrin glues,which were first described 40 years ago and are constitutedof concentrated fibrinogen (polymerization induced bythrombin and calcium) [1]. Nowadays, fibrin glues pre-paieU fiunt tiUmair^jlasma, such as Tisseel (Baxter,USA), are widely used. Autologous fibrin glues are con-sidered the best choice Lo avoid LuiilauiiiiaLlOh risk, buttheir use remains very I imited owing to the complexity andthe cost of their production protocols [2].

ConsequenTlyr'trie use of platelet concentrates toimprove healing and to replace fibrin glues, as firstdescribed by Whitman et al. [3], has been explored con-siderably during the last decade. Platelets contain high

Corresponding authoc Dohan Ehrenfest, D.M.' (LoB5srnac.com).

quantities of key growth factors, such as PDGF-AB (plate-let-derived growth factor AB), TGFb-1 (transforminggrowth factor b-1) and VEGF (vascular endothelial growthfactor), which are able to stimulate celt proliferation,matrix remodelling and angtogenesis, The use of thesegrowth factors to enhance healing is an interesting option,but commercial interests might obscure a lack of trueclinical benefits in some cases. Indeed, these concepts havespurred their commercial exploitation with the develop-ment of a wide range of preparation protocols, kits andcentrifuges. Most of these products were called PRP, thesame name as the original transfusion pfatefet concen-trates, which does not allow distinction between the differ-ent systems and protocols.

Ail available PRP techniques have some points in com-mon; blood is collected with anticoagulant just before orduring surgery and is immediately processed by centrifu-gation. The time for platelet concentrate preparation isvariable but is always completed within an hour. A firstcentrifugation step is designed to separate the blood intothree layers, red blood cells (RBCs) are found at thebottom, acellufaFplasma (PPP, platelet-poor plasma) isin the supernatant and a 'huffy mat' layer appears inbetween,irt which platelets are_mnmntrarftri (Figure IX1 Tie next steps vary among the numerous protocols but arean attempt to discard both the RBC layer and the PPP tocollect only the 'buffy coat' layer. Finally, the obtainedplatelet concentrate is applied to the surgical site with asyringe, together with thrombin and/or calcium chloride(or similar factors) to trigger platelet activation and fibrinpolymerization.

Choukroun's PRF (platelet-rich fibrin) is the latest de-vef6ptlUiMLof these protocols. Here, blood is collected with-out any anticoagulant and immediately centrifuged. Anatural coagulation process then occurs and allows forthe easy collection of a leucocyte- and platelet-rich fibrin(L-PRF) clot, without the need for any biochemical modi-fication of the blood, that is, no anticoagulants, thrombin oreatuuui Clihui tde are required. This open-access techniqueis the most 3impte"ana"aiso the least expensive protocoldeveloped so far. However, some confusion is likely becausedifferent suppliers use similar nomenclature for their dis-tinct products (such as Vtvostat PRF and Fibrinet Platelet-Rich Fibrin Matrix JPRFM]).

158 0167-7799/S - see front matter - 2008 Etsevter Ltd. AH rights reserved. dot:10.101B/|.tibtech.2008.11.009 Available online 31 January 2009

Review Trends in Biotechnology Vol.27 No.3

P-PRP — some PPP+ buffy coat fraction

X ' N

PPP

(discarded)

V. •/PPP

PPP(discarded)

L-PRP = some PPP

Step 2A. Option P-PRPHardspin long centrifugation

Step 2B. Option U-PRPHardspin long centrifugation

Step 1. Softspin short centrifugation

+ buffy coat+ residual RBC

TRENDS In Biotechnology

Figure 1. Classical manual platelet-rich plasma {PRP} protocol using a two-step centrifugation procedure [8,16]. Step 1: Whote Mood is collected with anticoagulants andbriefly centrifuged with tow forces (softspin). Three layers are obtained; red blood cells (RBCs), 'buffy coat* (BC) layer and platelet-poor plasma (PPP). BC is typically ofwhitish colour and contains the major proportion of the platelets and leucocytes. Step 2A: For production of pure PRP (P-PRP), PPP and superficial BC are transferred to^another tube. After hardspin centrifugation (at high centrifugal force), most of the PPP layer is discarded. I he ttnat P-PRP concentrate consists of an undetermined fractionoT Ut (OHftaining a large number of platelets) suspended in some fibrin-rich plasma. Most leucocytes are not collected. Step 2B: For production of leucocyte-rich PRP(L-PRP), PPP, the entire BC layer and some residual RBCs are transferred to another tube,~After hardspin centriffigaUori, the PPP is discardeo;. The final L-PRP consists of theentire BC, which contains most of the platelets and leucocytes, and residual RBCs suspended in some fibrin-rich plasma. Therefore, the final product greatly depends on themeans of SC collection. The transfer step is often performed with a syringe or pipette, with only eyeballing asmeasuring tool. Because the manual PRP process is not deartydefined, this protocol might randomly lead to P-PRP or L-PRP.

Definition of relevant parameters and classificationThree main sets of parameters are necessary for a clearclassification of platelet concentrates (Table 1), The firstset of parameters (A) relates to the preparation kits andcentrifuges used. The size of the centrifuge (parameter Al),the duration of the procedure (parameter A2) and the costof the device and kits (parameter A3) are significant factorswhen considering the repetitive use of these technicfues indaily surgical practice. The ergonomy of the kit and thecomplexity of the procedure (parameter A4) are also keyparameters because complex procedures are in danger ofbeing unusable or potentially misused, leading to irrepro-ducible results. For these reasons, automatized systemshave been developed and are commercially available.These parameters (A) define the practical characteristicsof each technique.

The second type of parameters (B) relates to the contentof the concentrate. The final volume of usable concentrate(parameter B1) depends on the initial blood harvest andcan define the potential clinical applications of a prep-aration protocol. The efficiency in collecting platelets(parameter B2) and leucocytes (parameter 83) and theirpreservation during the entire process (parameter B4)define the basic pharmacological relevance of ttie productand indicate its potential applications.

The third set of parameters (C) relates to thefibrin network that supports the platelet and leucocyteconcentrate during its application. The density of thefibrin network is mainly determined by the concentrationof the fibrinogen (parameter C1) during preparation [4].Most protocols lead to a low-density fibrin gel, whichallows for convenient surgical application but lacks atrue fibrin support matrix. By contrast, a high-densityfibrin network means that'The platelet concentratecan oe consioerea as a Diomateriat. and the fihrin matrixitSeii mignt nave potential healing effects [5], The fibrinpolymerization process (parameter C2) needs to be eval-uated, taking into account the ratios between fibrinogenand thrombin concentrations, as well as the faiomecha-nical properties of the final fibrin network. Fibrinogenis activated by thrombin, which initiates polymerizationintofibrin. However, the fibrin fibrillae can be assembledin two different biochemical architectures: either viacondensed tetramolecular or bilateral junctions or viaconnected trimoiecular or equilateral junctions [4]. Bilat-eral junctions are provoked by a drastic activation^and polymerization; foc_example witn rttqn tnrombtn'concentratJens, that leads to a dense network of mono-fibres similar to" a fibrin glue, which is not particularlyfavourable to cytokine enmeshment and cellular

159

Review Trends in Biotechnology Voi.27 No,3

Table 1. Definition of the key parameters to be evaluated in each platelet concentrate protocolKey parametersA: Preparation kits andcentrifuge(for processing of 50 mlof whole blood)

B: Platelets andleucocytes

C: Fibrin

Sub parametersA1: Size and weight of the centrifuge type requiredfor the method

A2: Duration of procedure {from bfood harvest tosurgical application)

A3: Cost (initial cost of equipment and repeatedcosts for reagents and kits)

A4: Ergonomy of the kit (including requiredmanipulations) and complexity of procedure

B1: Final volume of platelet get material (relative toinitial blood harvest)

B2: Platelet collection efficiencyB3: Leucocyte collection efficiency

B4: Preservation of the platelets and leucocytes

C1: Fibrinogen concentration and fibrin density

C2: Fibrin polymerization type

Definition• Heavy (and cumbersome)• Light (and compact)• Heavy but potentially light (i.e. a commercialized systemis heavy, but technique could be performed with a smallercentrifuge)• Quick, (less than 20 min)• Long (between 20 and 60 min)• Very long (more than 1 h)• Very inexpensive, less than 5 euros* Inexpensive, between 5 and 50 euros* Expensive, more than 50 euros• Very simple (+ +)• Simple (+)• Complex (« }« Very complex {• * )' Large, more than 25% of the blood sample• Small, less than 25%,* Variable, if additional fibrin-rich PPP can be preservedto increase volume above 25%• Excellent, more than 80%• Good, between 40 and 80%• Low, Jess than 40%• Sometimes unknown* No leucocytes, when technique eliminates most leucocytes- Healthy* Damaged* Unknown• Activated, when coagulation is induced during thecentrifugation process• High density• Low density• Strong, mainly trimolecular or equilateral junctions» Weak, mainly tetramoiecular or bilateral junctions

migration. On the contrary, a slow physiologicalfibrin polymerization yields a higher percentage of equi-lateral junctions, which allow the establishment of aflexible fibrin network with multifibre assembly that iscapable of supporting cytokine enmeshment and cellularmigration [6], Moreover, this organization will also pro-vide elasticity to the fibrin matrix comparable to that of asolid biomaterial. Fibrinogen collection efficiency andpolymerization type define the material characteristicsof the concentrate.

Using these sets of parameters, actual availablemethods can then be classified in four main categories,depending on the pharmacological (B parameters) andmaterial (C parameters) characteristics of the obtainedproduct (Table 2): pure PRP (P-PRP), leucocyte-rich PRP(L-PRP), pure PRF (P-PRF) and, finally, leucocyte-richPRF (L-PRF). In each category, the concentrate can beproduced by different processes (A parameters), either in afully automatized setup or by manual protocols.

Leucocyte-poor or pure platelet-rich plasma (P-PRP)Pure platelet concentrates for topical use were first devel-oped as an additional application of the classical transfu-sion platelet units and were first reported for maxillofacialsurgery [3,7]. ~ '

Automated protocols for P-PRP: plasmapheresis with alaboratory cell separator and Vivostat PRFThe first method of producing platelet concentrates fortopical use was the so-called plasmapheresis. whichTisesa lull sepafator,"elther in a discontinuous flnw <&t up (inwhich the patient stays connected to the machine and theblood filtering continues until the desired quantity ofplatelets has been collected) or starting from a bag ofharvested blood with anticoagulant [8]. These cell-separa-tor devices employ a differential ultracentrifugatipn(3000 g in general). The different Diood components, such

- as platelets, leucocytes and RBCs, are first separated fromthe PPP, which can then be re-infused to the patient. Whenthe integrated optical reader detects the firstc~b7jffy^elements" in Lhe stiiuut, these are automatically collectedinto a separate bag as the platelet concentrate (PRP). ASsoon as th& npf irai rparter ftetfirfls elements of RBCs; ptate-let collection is interrupted and RBCs, mixed with leuco-^cyteTand some residual platelets, are directed towards athird separateconection bag before eventual re-infusiorj.This method aHows~around 40 mL of PRP to be obtaineafrom 450 mL of whole blood. With discontinuous flow, inwhich the patient stays connected to the machine, up to300 mL of PRP could be collected [8], Despite the useof thissophisticated technology, the final PRP always contains

160

Review Trends in Biotechnology Vol.27 No.3

a.JDCD

CO

CO

O~O

2ga.

8-t-f

£'c<DOCo0

CD

2toa.

to

a>JC4-1tfc-

0c04J

g-

totoo

TJCtoen

<ACD

"oCO

COJCoOJ

3£

8ssBCO

to.EU

_C

I

IO

C•2cooof

«0

8IX

1 c^> s

rj "o <oU a. £i

"en

O Q

c•

toS

•* ££0 £X

ffl c

8 gS 3 8

c4J 5

CM J5 "gm Q. o

CDE

r^ "5CQ >

|>

OCo

< uj

4-f-- toen o< 0

C_o

.. 2CM 3< Q

<DOl

•g to

1 (an

d re

leva

nt R

efs)

T>j£

I

in

O _20- 0

CO

1

53

T3

.(0ESIQ

ifi

1u

o

c

sXUJ

'ra

CO

»

•

tu

cmcxXu

0)

^£>0}

rOJX

CO

Cel

l sep

arat

or P

RP

[

IDQJ

S3

-Q

S

TOD

S

oo3

o

I

(0

ECO

-i-

CO

"tocQ)Q.

ui

O3tr3

(D(U

S

01a.

o

a.

ED

1

So

c

occ3

l/l

1oo

o

1_ffl

.ECO

•

CD

CCDa.X

£c

D)

°

£

sijTl CO

J>£

II

'

3

£ °-« O-

§2

< 5Q_

O.o;

2:

CD

1

So

c

cc

•aoo

13

§

O

0}

(Q

>

+

CD

"toCCDCLXUJ

B)c3

caCDX

co1

CO

* oT05 °- Q: 3a. a. £i

CL CD ^ — 2: a c Q-0- Q. .2 QL

<3 I |=OT0 E ,2 a.Q- t/) ^ O

Q.

CD

1

?

°

C

Occ3

T3OO

T3OO

CO

JSta

>

•

CD

"to

CDQ.

XI

a)co

aj

CO !>CD oX a.

Fria

dent

PR

P [

16]

Cur

asan

PR

P [8

]R

egen

PRP

Pla

telte

x PR

P [1

7]Ac

e PR

P [1

5]

o.

Q.cdQ_

03coi;co

_c

X

-oCOc3

^oCO

1tcffl •X

(A

|

3_CO

0

•DOOO

CB

td

+

ID

"w

Q.Xm

D)C

3^

siCD

CO oX Q.

Flbr

inet

PR

FM [9

]

Q-

O.cL

?Oi

jr

X

_

ro

-^to

f15<DX

Eo

c

o

O)ro_j

+

+

S>"t/iQ)D.XCD-£

CD>

O

O

a

.cO)_J

0enI

Cho

ukro

un's

PR

F [z

Q.

u.QLa.

s1fDc:

01

sasa.

2

Soa.CD

CD

E

ta

S2oa.

"3(O

oT

c

"5>£

residual RBCs and leucocytes. In addition, this protocol iscumbersome and labour-intensive and often requires thehelp of haematoJogist. Although it is the more accuratemethod from a technical point of view 18], its use in daiJypractice remains rare. Recently, more compact systemshave been developed that can be used more easily, bothfor autotransfusion during surgery and for topical appli-cation (for exampie the Elects eetl separator, Sorin group,Italy).

The Vivostat PRF centrifuge (Vivolution. Denmark)can be considered as an advanced ceil separator, and itwas originally designed to produce the Vivostat Fibrinsealant. The use of a specific preparation kit with thiscentrifuge allows the production of a leucocyte-poor pla-telet concentrate for surgical use. However, VivostatPRF has been used in only a few published *stu3Tes,and this system is cumbersome and very expensive fordaily practice. Moreover, its platelet collection efficiencyis rather low and platelets are damaged ounncpErieprocess [9],

Manual protocols for modified P-PRP: Anitua's PRGFOne of the first piatefet concentrate protocols (PRGF,which stands for either plasma rich in growth factors[10] or preparation rich in growth factors [11]) wasdescribed in 1999 by Anitua and has been commercializedfay BTl (BioTechnoiogy institute, Vitoria, Spain). In thisprotocol, venous blood is collected and centrifuged In sevT

era! small tubes to obtain the three typical layers; RBCs,'buffy coat' and aceHular plasma. The upper part of theacellular plasma is called plasma poor in growth factors(PFGt-j and is ach -pipet-

avoid creating turbulences. The remaining plasmais termed PRGF and is collected with a pipette, using only'eyeballing* as a measuring tool (Figure 2). Several pipetirigsteps, each associated with possible pipeting and handlingerrors, are necessary to collect the entire PRGF fraction ofthe patient, afte£which fibrin polymerization is induced bya 10% calcium chloride solution. ^After 15 to ZQmtn, anunstable PRGJL{jftl is fnrmfyi that will need to b_e usedimmediately. _

Trierearesome inconsistencies in the PRGF protocol. Inthe original description of the protocol [10], most of theplasma (after discarding a small fraction as describedabove) was collected, including the 'buffy coat' layer thatcontains most of the platelets and leucocytes. However, inlater applications of this method [12,13], the authors claimthat the buffy coat layerwas not collected. The objective ofthis approach was to avoid the collection of leucocytes, butit seems_tp_be technically imprecise and in danger _ofyielding irreproducible results. Moreover, it also leads toaTow_oJajelet collection efficiency pecause platelets andleucocytes are found together in the intermedia^after lowspin centrifugation 114 Platelet preservation hasnot been examined, but the soft centrtfuqation should keepplatelets in gop^_sfe^r"Anitua's PRGF method is aninexpensive manuaFprotocof for the preparation of leuco-cyte-poor PRP. However, the lack of ergonomy and repro-ducibility of the procedure is problematic, Other similarprotocols can now be found in the literature, for exampletheNahitaPRP[15],

761

Reviewpv

PPGF(1mL)

PRGF(1mL)

Step 2. Pipetting

TRENDS In Biotechnology

Figure 2. Commonly described protocol for Anitua's PfifiF [10-14]. Step 1: CftratedWood {in S mL tubes) Is softly centrifuged {B mln at 460 g) and separated into threelayers; RBC base, buffy coat (BC) and aceliufer plasma. Aceilutar plasma containsthe empiricaity defined layers plasma poor in growth factors (PPGF) and plasmarich in growth factors (PRGF). Step 2: The PPGF layer (1 mO is discarded, and thePRGF, just above the BC, is collected by careful pipetting. PRGF from alt sampletubes is collected into one tube and calcium chloride is added for clotting.

Leucocyte- and platelet-rich plasma (L-PRP)The initial objective of developing alternative easy-to-handle methods was to make it possible to use plateletconcentrates in daily practice without having the supportof a transfusion laboratory. Without a celt separator, elim-i nation of leucocytes becomesmore difficult, and the result-ing platelet concentrates therefore contain a high quantityof leucocytes, which were not initially desired. However,through changes in the collection parameters, the sameprotocols might also be used to produce pure leucocyte-freePRP.

Manual protocols for L-PRP: Curasan, Friadent-SchOtze,Regen and PtatettexTwo similar protocols, each using a two-step centrifugationprocedure, were marketed by Curasan (Kleinostheim,Germany) [8] and Friadent-SchGtze (Vienna, Austria)[1 &], respectively. Each method follows the first principalstep described above and shown in Figure 1, in which a firstcentrifugation step separates the blood components intothree layers of RBCs, T>uffy coat' and PPP. The PPP andbuffy coat 1 ayers are then carefully collected, avoiding RBCcontamination, and transferred to another tube, wherethey are subjected to a second centrifugation step at highspeed, which separates the sample again into its com-ponents. After the second centrifugation step, most ofthe PPP layer is discarded using the "eyebaf ling' method.The PRP concentrate obtained with this method is com-posed of a high quantity of platelets, leucocytes and circu-lating fibrinogen, but it also contains residual RBCs. Theconcentrate is applied with bovine thrombin and calciumchloride.

Trends in Biotechnology Vol.27 No.3

Similar methods for PRP generation have also beendeveloped with Plateltex (Bratislava, Slovakia) and RegenPRP (Regen Laboratory, Moliens, Switzerland). The Pla-teltex protocol uses specific gelifying agents, such ascalcium ojuconate and lyophtltzed purified batroxobin,an enzyme that cleaves fifartnopppj-irtA, tn induce fibrinpolymerization without bovine thromfain and gel lino inground 10 mm [17j, The Regen method employs a separa-Sr gei within the centrifugation tubes with the aim ofimproving the collection of platelets and leucocytes.

All these protocols require substantial manual pro-cedures, meaning that the preparation process is time-consuming, and furthermore, they only lead to smallvolumes of L-PRP. Some of the PPP fraction can be pre-served: it contains fibrinogen and al lows an increase in thefinal L-PRP volume. Adapted kits can be quite expensive ifused frequently. The final product exhibits a low densityfibrin matrix, which is strong enough for application as afibrin glue but quickly dissolves. Platelets and leucocytesare typically well preserved and concentrated in theseprotocols,, but the success of the method depends on theoperator and results are not always reliably reproduced.Numerous modifications of this basic protocol with regardto centrifugation forces and time and the type of antic-oagulants have been used in different studies or commer-cialized (for example. Ace PRP) [15], In most publicationson these techniques, the composition of the final PRPs usedis often unclear. For example, if the buffy coat layer is notcompletely collected, the platelet collection efficiencydecreases, and P-PRP can sometimes be produced insteadof L-PRP (Figure 1).

Automated protocols for L-PRP: SmartPReP, PCCS, GPSand MagellanAutomated systems for L-PRP have been developed in theform of PCCS (Platelet Concentrate Collection System) by31 (Palm Beach Gardens, USA) [9,14] and SmartPReP byHarvest Corp (Plymouth, USA) [9,16]. The centrifugesused have been designed to take a customized collectionand centrifugation device, which consists of two connectedcompartments. In the PCCS method, citrated whole bloodis transferred into the first compartment and centrifugedbriefly to obtain the three layers (RBC, buffy coat, PPP).Then, by opening of a tubule and using air pressure, thesuperficial layers (i.e. PPP and buffy coat) are transferredto, the second chamber and centrifuged again but for alonger period. Finally, using the same air pressure system,most of the PPP layer is transferred back into the firstcompartment and thus discarded. The final product Is richin leucocytes and has similar characteristics to the manualCurasan PRP described above.

The SmartPReP protocol requires even less manipula-tion. The two-chamber device is designed to automaticallytransfer the upper layers (PPP and buffy coat) into thesecond chamber based on variations in weight andcentrifugation speed. SmartPReP is a multifunction sys-tem [18]: using a specific collection and separation kit, thiscentrifuge can also be used to concentrate stem ceils frombone marrow aspirates.

The Magellan APS (Autologous Platelet Separator) byMedtronic (Minneapolis, USA) can be considered as a

162

Review Trends in Biotechnology Vol.27 No.3

further advance of the previously described ceil separatorwith optical reader. However this device is compact,similar to a table centrifuge, and designed for smai? bloodsamples of up to 50 mL. The obtained platelet collectionefficiency is high, but cell preservation is unknown [19].Data sheets provided by the company indicate that theleucocyte content is also high.

GPS (Gravitational Platelet Separation System) by Bio-met Biologic (Warsaw, USA) is another variation of a two-chamber centrifugation device with two-step centrifu-gation protocol [20]. The main difference is that the PPPis discarded after the first centrifugation using a syringeand tubutes, and the second centrifugation step is per-formed with the RBC layer. The final PRP concentrate iscol tected by aspiration of the buffy coat layer on the surfaceof the RBC base. The procedure is thus inversed, but thefinal result seems to be similar.

The main drawbacks of all these techniques are thatthey require expensive and cumbersome centrifuges andcollection/preparation kits. Moreover, the final concen-trates dissolve quickly, similar to a fibrin glue. Their usein daily practice remains uncommon and the PCCS is nolonger available.

Leucocyte-poor or pure platelet-rich fibrin (P-PRF)concentratesIn this category, there is only one method available. TheFibrinet PRFM kit by Cascade Medical (New Jersey, USA)col Hdli is two Lubes, ut ie fui blood collection and another forPRFM dotting, together with a transfer device. A smallamount of blood (typically 9 mL) is drawn into a collectiontube, which contains tri-sodium citrate as an anticoagulantand a proprietary separator gel, and centrituged for sfxminutes aL f tlyh speed. \e three typical layers of RBCs,burryTrualand PHP are obtained. Buffy coat and PPP areeasily transferred to a second tube containing CaC!2 withthe help of a specifically designed tube connection system.The clotting process is triggered by the presence of CaCI2

and the tube is immediately cerrtrifuged for 15 min, after

which a stable PRFM clot can be collected. The companyclaims that the sysjSBrpr-edyces a 'natural' platelet con-centrate owing to^the absenoTof^bovine thrombin. How-ever, this claim is\doubtfu} becausethe blood is mixed withanticoagulant and separation el, leading to what could beconsidered unnaturalasndtdons.

This protocol is similar to other typical L-PRP proto-cols, such as the Curasan method. Thema in difference isthat only very low amounts of leucocytes are collected

U> Uie sufecmc separator gel used in the method.However, the platelet collection efficiency is high and thepreservation of the platelets during the procedure seemsto be acceptable [9]. Platelet activation and fibrinpolymerization are triggered using only calcium chloride,the same method as in Anitua's PRGF protocol. However,the fibrin matrix in FJbrinet PRFM is denser and morestable than that in PRPs, probably due to the dynamicclotting during the second centrifugation step, which ismore efficient than a static PRP polymerization. Thesimultaneous processing of large sample numbers withthe Fibrinet method remains difficult and is expensive indaily practice. In addition, fundamental or clinical stu-dies demonstrating the efficiency of Fibrinet PRFM arenot yet available.

Leucocyte- and platelet-rich fibrin (L-PRF) concentrates:Choukroun's PRF ^c \~U^Choukroun's PRF protocol is a simple and free techniquedeveloped in France by Choukroun et at. [21]. It can beconsidered as a second-generation platelet concentratebecause the natural concentrate is produced without anyanticoagulants or gelriymg agents [22]. Venous blood iscollected in dry glass tubes and centrifuged at low speed(Process protocol, Nice, France) [23]._ in the absence ofanticoagulants, platelet activation and fibrin polymeriz-ation are triggered immediately. Therefore, after centrifu-gation, three layers are formed: the RBC base layer,acellular plasma top layer and a PRF clot in the middle(Figure 3). The PRF clot forms a strong fibrin matrix with a

TRENDS in Biotechnology

Figure 3. Choukroun's platelet-rich fibrin {PRF} method [21-303. {a} Blood is softly cerrtrifuged Immediately after collection without anticoagulants, and coagulation startsquickly. Blood is separated into three components with the formation of a strong fibrin clot in the middle of the tube. This ctot acts as a plug that traps most light bloodcomponents, such as platelets and leucocytes, as wett as circulating molecules, such as growth factors and flbroneetin. This method leads to the natural production of adense leucocyte-rich PRF {L-PRF) dot. {b) After compression of the L-PRF cfot, it can be used easily as a membrane (actual length shown: 3 to 4 cm).

163

Review Trends in Biote.cnn.ology Vbl.27 N.o.3

^architecture, in jfttfiich most ofthe platelets and leucocytes from the harvested biood areconcentrated [24,25]. When pressed between two' gauzes,the PRF clot becomes a strong memhjranfi. J)nrt some appf i-jqajEloas fit t Ms MogojLis Momateriaf have been describedin oral [26], maxiftofatial [27,28], ENT (ear, nose, -throat)[29] and plastic surgery [30].

Unlike the PRPs, Choukrouris PRF floes -not dissolve

is -slowly remodelled in a similar way to a natural bloodclotr-PtetDlots .and leucocytes are uilleUtoll wdlt high•efficiency Tn this -method -Sftd leticoeytes are -preservedthroughput. However, .platelets are activated during ingprocess, which leads to a -substantial embetkiiftq of plate-let and leucocyte growth factors into the fibrin matrix[24,25].

This method allows the production of a high quantity ofL-PRF clots simultaneously using either a specific centri-fuge that takes eight tubes or .any modified laboratoryCentrifuge, making it possible to produce -even more clotsfor larger surgeries. Another advantage of this method isits tow cost and the great ease of the procedure, whichallows the production of many concentrates quickly and bynatural means, that is, wfthoat tiie -use of chemicals -orunnatural conditions. Therefore, this method seems to bemost suitable for widespread usein daily Practice arffl isactually tne main tecnntque in some countries, includingfra'nce, lialy atiil lU'ael. £JM'-lii should -be .undertaken'tofurther develop trttS protocol -m the near future.

P-PRP or L-PRP? Potential applications andpoatnoyjersies-IVlost studies involving the use -of PRPs -have employeddifferent irnhouse protocols in which the basic two-step

gallon forces {from 160 g to 3000 g) and time (from 3 toZdrrrin for the first centrifugation step). The definition ofthese parameters frequently .seems to Jae empirical andcross-examination of these technical data is an impasse.Moreover, it is very difficult to judge whether the actualexperiments have been performed with P-PRP or -withL-PRP [31 ]. P-PRPs seem to be the most frequently testedplatelet concentrates in vitro and m vivo, but an accurateanalysis of the entire literature is impossible,

The first published in -vitro studies demonstrated ageneral tendency of PRPs to stimulate the pro!Iteration-of several cell types, including osteoblasts [32,33], ftiro-blasts [34], tendon cells [35], chondrocytes [36], periodontaitigafnent cslts [37] and bone mesencftymed -stem cells(BMSGs) [38]- However, contrasting results haveaJso beenreported [39,40], and this topic is still hotly debated in theliterature, probably owing to the great number of PRPprotocots that might tead to significantly different pfelefetconcentrates. Jn addition, experiments were often per-formed with animal cell tines [41,42] or commercializedcell lineages [40,43], even though primary cultures ofhuman -celte [33,39,44] should have been used as :ttte taldstandard because of the high immunogenicity of any plate^letconcentrate, which will always contain some leucocytes?The effect of PRPs in differentiation is aiso controversialbecause some studies demonstrated a stimulation of osteo-

blastic differentiation [32,33,42], whereas others reportedan inhibitory effect [39,41] " ' ^

"vriro stimulations provoked by platelet concentratepreparation are orily Observed for a short ti'rne, which hasled to discussion over their finical long-term significance[45] because most authors consider -them as being onlyeffective in vivo over a short period [Afi] Jr)p-hp<rt pmiifor.atiorj results in vitro:have:been otftained with physitftogfca'lplatelet amougts, that js, far a PRP witfe acgngaTton tnat was equivalent to 2.5times the bloodconcentration [44], Hiotter pffitplptto mduce-rrtetiativfe -effects. ;However, trie-data on thfe dbse-dependenj effects -are lOTted^nd shftuld ;te .inyesjigajedf urthef after the fibrin and leucocyte content of the platelet-conceatrate has been accurately defined.

'Clinical studies have indicated that platelet gets "canshorten recovery time, ;red.uce:surgery-relajgd^s«weii4ngandpain [47], accelerate the repair of the soft tissues [48] andincrease bone regeneration in the short-term [45], How-wer, allthese benefits eoijM also be observed wfth the useof simpS fibrin gktes [2,18], in most cases, PKPs areconsidered and usea as a sephisticated autotogous fibringlue^ and the literature remains uncJear .about the fruectinica! differences -between -these related fibrin proifects.

With regard to the use of platelet concentrates in bio-technological applications, it is worth noting that promis-ing results have already been obtained for bone tissueengineering studies. BMSCs. collected from .the iliac-crest"were treated with PRPs -as-a-eeli culture and implantationmedium [49] with the aim of replacing fetal calf serum inretmptanted materials to avoid contamination or immuneresponse [50]. PRPs^w^ce^shown to strong I v stimulate-proiiferatiorrof BlvlS-Cs i>ot -jnr"hfrpf*'1'h':'1'r -[38,51], This .method for tissue engineering with BMSGsand PRP might be relevant in reconstructive cramofacialsurgery [52] or in orthopaedics [53]. These invasive proto-cols -were tested in human periodontai [54] and implantsurgery [551, although .they seem to be currently imprac-tical for use in daily practice.

Most studies employing Anitua's PRGF (manualP-PRP) -were performed ;by BTf, the tlentat implantcompany £OHimerciaJizing th.is product. .Preliminaryresults on chronic ulcers have been encouraging but werealso limited because only small wounds were treated,-which did not entirely close {12]. Wider appiications-of thisconcentrate type in orthopaedic surgery, for example .OBtendons, has been proposed [13] but will need to be vali-dated independently.

TMmatndifferencesMtweenttetypes of .platelet concentrates are related to their leucocytecontent and the potential effects of the leucocytes onproliferation, differentiation, immunity and infection,Otie to the great iTariabilfty in the different protocols,the obtained results have rffiser-been-analy2ed~usintg theleucocyte content of the final concentrate as a keyparameter. Thus, differences between P-PRP and L-PRPpreparations_teya ft"* yfet fa**"*1 fttsm-at-ely docutfierited.

r~" '~"

Leucocytes and fibrin: the two key parametersThe literature dealing with platelet concentrates oftenignores the impact of leucocytes and fibrin, which are

164

Review

the two Jcey parameters in our classification ^Figure 4).Some authors even recommend, without any scientificevidence, the elimination of leucocytes [11]. However, sev-eral studies nave already pofntMottt the~key roTe"^ofleucocytes Joi-M-1 [3'lj, Jbototortheiranti-iflfectJousactioo'[56,57J ana immune regulation [25,58]. Apart from an anti-infectious effect, leucocytes "produce large amounts ofVEGF [59], Platelets are known to contain angiogenesisstimulators, such as VEGF and basic ftbroblast growthfactor, and inhibitors, such as endostatin and thrombos-pondin-1, in similar quantities [60]. Additional VEGF inPRP preparation, -which stems from the leucocytes, mightbe crucially important for the promotion of angiogenesis,

When used during coronary artery bypass surgery,L-PRP significantly reduced occurrences of chest woundinfection, chest <Jrainage and teg wound -drainage [61 ]. Theleucocyte content did not seem to induce negative effects orto impair the potentially beneficial effects of PRP, evenwhen used in joints [62], Recent research showed~tnatQPRP was afafiT^rstifftalate anabofem and

Trends in Biotechnology Vbt27 No.3

Injected form for the treatment of teadonJtis [64]. injectedL-^Kt3 could also be used TOT tTffi'u^dlntent of long bonedelayed healing [65], An uncontrolled immune reaction ofL-PRPs has ftot been f eported and, trti the contrary, theiruse was able to dintinisfa pain and Jnflammatioji of thetreated sites [62,64]. However, the respective effects ofplatelets and leucocytes in the platelet concentrates havenot yet been analyzed, aftd the contribtitfon of the -leuco-cytes to the observed overall effect remains unclear,altimtqhsynerqistic fiffp.c1;s can he

capacities of tendons [63j and could be successfully used in

need to focus on this aspect of PRP func-tion to tfearfy elucidate the relative contributions -of tSieconcentrate componeots, thereby Jielping to ^develop «a>Centrates with specific and desired effects.

The density of the fibrin matrix and its composition isanother key parameter of any platetet -concentrate [43].However, most studies addressing the biological effectsof platelet concentrates focused on investigating plateletgrowth factors and ignored, the cytokines in theirenvironment w the influence of the fibrin matrix, whichsupports their release. The fibrinogen concentration

7HHWDS in Biotechnology

-Figure*. Schematic iflusttation of the matrixandcett architecture of thefour categories of plateJetconcentrates-TT^rokeyparametere are importar^Meococyte content (bluecircles) and denstty of TIbrta tyeflowflightbrown fibres). Platelet aggregates XUght-grey shapes) are always asserrtbted on Che Ttbrln fibres. In 'typical P-PRP and T.-PRPpreparations (top panels), the fibrin network is immature and consists mainly of fibrillae with a small diameter (red .arrows) due to simple fibre polymerization. This fibrinnetwork supports platelet application during surgery but dissolves quickly like a fibrin gJue, In P-PRF and L-PRF preparations (bottom panels), fibrin fibres are thick (blackarrows) due to muftlpte fibre assembly and constitute a resistant matrix that cart be considered as a fibrin otomaterta!.

165

Review Trends in Biotechnology Vol,27 No.3

varies considerably between the different methods,- forexample, in P-PRP protocols, the fibrinogen mainiyoriginates from the platelet a granules after activationand the final fibrin concentration is low, whereas in otherprotocols, circulating fibrinogen is also collected andreinforces the final fibrin network, Piateiet concentratesshould be analyzed as a whole, as an assembly of plate-lets and leucocytes in a complex fibrin matrix. Plateletgrowth factors cannot be dRsrrihpf1motecuies and should be consideredbiology puiia uf vitw

ConclusionsThe world of platelet concentrates for surgical use isactually a jungle of commercial proposals and unclearproducts. Under the same name, more thari

autolocjousTjlutts w blumaLbi ialiTare available. The tech-^areayaunological classification pressntecfriere^imed to provide anoverview of the available systems and to categorize themwith respect to three main parameters: fibrin density,leucocyte content and degree of standarization of the pro-cedure. PRPs are often considered as improved fibrin glues;however, PRFs can be regarded as dense fibrin biomaterialwith biomechanicaf properties. A high density fibrin clotcan serve as a biological healing matrix by supporting cellmigration and cytokine release, expanding the range of itspotential applications greatly. The influence of the leuco-cytes on the biology of each product and its potentialbenefits shouid now be carefully analyzed because it couldexplain many controversial data from the literature.'Finally, expensive and complex procedures are often unu-sable in daily practice and many will disappear. Simpleand free systems, such as Choukroun's PRF, were devel-oped by clinicians for clinicians and are anticipated to bemajor methods in the next years.

We fee! that clarification is the first step in defining anyclinical and faiotechnological applications for each tech-nique, and the development of these products is nowcompletely dependent on an accurate and rational descrip-tion of their structure and associated biology.

AcknowledgementsThis work was partially supported by a grant from the LoBS Foundationfor Research, AP-HP, Paris, France. The authors declare no competingfinancial interests.

References1 Matras, H. (1970) Die Wirkungen vershiedener Fibrinpraparate auf

Kontinuftat-strennungen der Rattenhaut, Osterr. Z, Stomatol. 67,338-359

2 Gibfafe, J.W. and Ness, P.M. (1990) Fibrin glue: the perfect operativesealant? Transfusion 30, 741-747

3 Whitman, D.H. et al. (1997) Platelet gel: an autologous alternative to-fibrin giue with applications in oral and maxifloferaa} surgery. J. OralMaxillofac. Surg. 55,1294-1299

4 Mosesson, M.W. et al. (2001) The structure and biological features offibrinogen and fibrin. Ann. N. Y. Acad. Sci. 936,11-30

5 Clark, R.A, #001) Fibrin and wound healing. Ann, N. Y, Acad, Sci, 936,355-367

6 van Hinsbergh, V.W. et al. (2001) Role of fibrin matrix in angiogenesis,Ann. N. Y. Acad, Sci, 936, 426-437

7 Marx, R.E. et al. (1998) Platelet-rich plasma: growth factorerftiancement for bone grafts. Orai Surg. Oral Med. Oral Pattioi.Oral Radio). Ended. 85, 638-646

8 Weibrich, G, etal. (2003) Comparison ofplatelet, leukocyte, and .growthfactor levels in point-of-care platelet-enriched plasma, prepared usinga modified Curasao kit, :ssiyj preparations received from a local fetoodbank. Clin. Oral Implants Res. 14, 357-362

9 Leitner, G.C. et a!. (2006) Platelet content and growth factor release inplatetet-rich plasma: a comparison of four different systems. Vox Sang.91, 135-139

10 Anitua, E. (1999) Plasma rich in growth factors: preliminary results ofuse in the preparation of future sites for implants. Int. J. OralMaxilfofec-Jfl

Tet a). (2007) The potential impact of the preparationfactors (PRGF) in different medical

4551-456012 Anitua, E. et al. (2008) Effectiveness of autologous preparation rich in

growth factors for the treatment of chronic cutaneous ulcers. J.Btomed. Mater. Res. B Appl. Bromster. M, 415-421

13 Sanchez, M. et al. (2007) Comparison of surgically repaired Achillestendon tears using platelet-rich fibrin matrices. Am. -I. Sports Med, 35,245-251

14 Weibrich, G. et ai. (2005) Comparison of the platelet concentratecollection system with the ptasma-rich-m-growtlvfactors kit toproduce platelet-rich plasma: a technical report Int J. OralMaxitlofac. Implants 20,118-123

15 Tamimi, F.M. et a). (2007) A comparative study of two methods forobtaining platelet-rich plasma. J. Oral Maxillofac. Surg. 65,1084-1093

16 Weibrich, G. et al. (2003) The Harvest Smart PRePTM system versusthe Friadent-Schutze platelet-rich plasma kit, Clin. Oral Implants Res,14, 233-239

17 Mazzucco, L. et al, (2008) Platelet-rich plasma and platelet gelpreparation using Plateltex, Vox Sang. 94, 202-208

18 Mart, D. et a}. (2001) The use of autologous platelet-rich plasma(platelet gei) and autologous platelet-poor plasma (fibrin glue) incosmetic surgery. Plast ftecortstr. Surg. 1O7, 229-237

19 Christensen, K. eta!. (2006) Autologous platelet gel: an in vitro analysisof platelet-rich plasma using multiple cycles. J, Extra Corpor. Techno!,38, 249-253

20 Martovits, S, «t al, (2004) A new simplified technique for producingpiatelet-rich plasma: a short technical note. Eur. Spine J, 13 (Suppt, 1),S102-S106

21 Choukroun, J. et al. (2001) Une opportunity en paro-implantologie: lePRF. Implantodontie 42, 55-62

22 Dohan, D.M, et al. (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts andevolution. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod101, e37-e44

23 Dohan, D.M. et al. (20O7) Cytotoxicity analyses of Choukroun's PRF(Platelet Rich Fibrin) on a wide range of human cells: the answer to acommercial controversy. Oral Surg. Oral Med. Oral Pathot. OralRadiol. Endod. 103, 587-593

24 Oohan, O.M. et al, <2006) Ptobelet-ricri fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologicfeatures. Oral Surg. Oral Med. Orai Pathol. Oral Radiol. Endod.101, e45-e50

25 Dohan, D.M, et al. (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part 111: leucocyte activation: a newfeature for platelet concentrates?. Oral Surg. Oral Med. Oral Pathol.Oral Radiol. Endod. 101, e5i-e55

26 Choukroun, J. et al. (2006) Platetet-rich fibrin (PRF): a second-generation platelet concentrate. Part 1V: clinical eflBcli on tissaeheating. Oral Surg. Oral Med. Oral Pathol. Ora! Radio!. Enciod.101, e56-e60

27 Choukroun, J. et al. (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: htstologic evaluations ofPRF effects on bone allograft maturation in sinus lift Orai Surg.Oral Med. Oral Pathol, Orai Radiol. Endod. 101, 299-303

28 Diss, A. et al. (2008) Ostootome sinus floor elevation using Choukroun'splatelet-rich fibrin as grafting material: a one-^ear prospective pilotstudy with microthreaded implants, Oral Surg, Oral Med. Oral Pathol.Oral Radiol. Endod. 105, 572-579

29 Choukroun, J.I. et at. (2007) Influence of platelet rich fibrin (PRF) onproliferation of human preadipocytes and tympanic keratinocytes: anew opportunity in facial (fpostructure (Coleman's technique) ancftympanoplasty? Rev. taryngol, Otol. Rhinoi. (Bord.) 128, 27-32

Review Trends in Biotechnology Vol.27 No,3

30 BraceinL F, and Dohan, DM. (2007) The relevance of Choukroun'splatelet rich fforin (PRF) during facia} aesthetic lipostructure<Cofeman's techniqcie); preMfnrnary results. Rev. t-aryngol. Qtol.Rhinoi. (Bord.) 128, 255-260

31 Everts, P.A. «t al. (2008) What do we use; platelet-rich plasma orplatelet-leukocyte gel? J. Biomed. Mater. Res, A 85,1135-1136

32 Clausen, C. et al. (2006) Homologous activated platelets stimulatedffferentiation and proliferation of primary human bone cells. CellsTissues Organs 184, 68-75

33 Uggeri, J. et al. (2007) Dose-dependent effects of platelet get releasateon activities of human osteoblasts. J. Periodontol. 78,1985-1991

34 Krasna, M. et al. (2007) Platelet gel stimulates proliferation of humandermal fibroblasts in vitro. Acta Dermatovenerof, Alp, Panonica Adriat,16,105-110

35 Anitua, E. et al. (2005) Autoiogous preparations rich in growth factors• promote proliferation and induce- VEGF and HGF production by

human tendon cells in culture. J. Orthop. Res. 23,281-28636 Akeda, K. et al. (2006) Platelet-rich plasma stimulates porcine

articular chondrocyte proliferation and matrix biosynthesis.£)steoarthrttis Cartilage 14,1272-1280

37 Okuda, K. et al. (2003) Platelet-rich plasma contains high levels ofplatelet-derived growth factor and transforming growth factor-b andmodulates the proliferation of periodontaliy related cells in vitro. J.Periodontol. 74, 849-857

38 tucarelli, E. et al. (2003) Platelet-derived growth factors enhanceproliferation of human stroma! stem cells. Biomateriats 24, 3095-3100

39 Cenni, E, et al. (2005) Effects of activated platelet concentrates on•human primary cultures of fibrofafasts and osteofalasts, J. Periodontol.76, 323-328

40 Slapnicka, jv et at. (2008) Effects of activated and nonactivatedplatelet-rich plasma on proliferation of human osteoblasts in vitro.J. Oral Maxtltofac. Surg. 66, 297-301

41 Soffer, E. et al. (2004) Effects of platelet lysates on select bone cellfunctions. Ctia Oral Implants Res. 15, 581-588

42 Goto, H. etal. (2006) Platelet-rich plasma/osteoblasts complex inducesbone formation via osteoblastic differentiation following subcutaneoustransplantation. J, Periodontai Res. 41, 455-462

43 Kawase, T. et af. (2003) Platelet-rich plasma-derived fibrin dotformation stimulates collagen synthesis in periodontal ligament andosteoblastic ceils in vitro. J. Periodontol, 74, 858-864

44 Graziani, F. et at. (2006) The in vitro effect of different PRPconcentrations on osteoblasts and fibroblasts, Clin. Oral ImplantsRes. 17, 212-219

45 Thor, A. et al. (2007) Early bone formation in human bone graftstreated with platelet-rich plasma: preliminary histornorphometricresults. Int. J. Oral Maxitlofac, Surg. 36,1164-1171

46 Piachokova, A.S. et al. (2008) Effect of platelet-rich plasma on boneregeneration in dentistry: a systematic review. Clin. Oral ImplantsRes. 19, 539-545

47 Everts, PA. et a!. (2007) Autoiogous platelet gel and fibrin sealantenhance the efficacy of total knee atihroplasty: improved range ofmotion, decreased length of stay and a reduced incidence ofarthrofibrosis. Knee Surg. Sports Traumatol. Arthrosc. 15, 888-894

48 Lindeboom, J.A. et al, (2007) Influence of the application of platelet-enriched plasma in oral mucosal wound healing. Clin. Oral ImplantsRes. IS, 133-139

49 Yamada, Y. et al. (2004) Autogenous injectable bone for regenerationwith mesenchyma! stem cells and platelet-rich plasma: tissue-engineered bone regeneration. Tissue Eng. 10,955-964

50 Ooucet, C. et al. (2005) Platelet lysates promote mesenchymai stem cellexpansion: a safety substitute for animal serum in cell-based therapyapplications. J. Celt. Physiol. 205,228-236

51 Arpornmaeklong, P. et al. (2004) Influence of platelet-rich plasma(PRP) on osteogenic differentiation of rat bone marrow stromal cells.An in vitro study, int. J. Oral Maxillofac. Surg. 33, 60-70

52 Hibi, H, et al. (2006) Alveolar cleft osteoplasty using tissue-engineeredosteogenic material. Int. J. Oral Maxillofac. Surg. 35, 551-555

53 Kitoh, H, et al. (2007) Transplantation of culture expanded bonemarrow cells and platelet rich plasma in distraction Bsteogenesis irfthe long bones. Bone 40, 522-528

54 Yamada, Y. et ai. (2006) A novel approach to periodontal tissueregeneration with mesenchymat stem cells and platelet-rich plasmausing tissue engineering technology: a clinical case report. Int. J,Perioclontics Restorative Dent, 26, 363-369

55 Ueda, M, et al. (2005) Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneousimplant placement. Int. J. Periodontics Restorative Dent. 25,129-137

56 Cieslik-Bielecka, A et at (2007) Why the platelet-rich get hasantimicrobial activity? Oral, Surg, Oral Med. Oral Pathol. OralRadiol. Endod. 103, 303-305

57 Moojen, DJ. et al. (2008) Antimicrobial activity of platelet-leukocytegel against Staphylococcus aureus. J. Orthop. Res. 26, 404-410 ,

58 Et-Sharkawy, H, etat, (2007) Platelet-rich plasma: growth factors andpro- and anti-inflammatory properties. J. Periodontal. 78,661-669

59 Werther, K. et al. (2002) Determination of vascular endothelialgrowth factor (VEGF) in circulating blood: significance of VEGF mvarious leucocytes and platelets. Seand. J. Clin. Lab. Invest 62,343-350

60 Italiano, J.E., Jr et al. (ZOOS) Angiogenesis is regulated by a novelmechanism; pro- and antiangiogenic proteins are organized intoseparate platelet alpha granules and differentially released. Blood111,1227-1233

61 Khatafi, R.S. et al. (2008) Topical application of autologous bloodproducts during surgical elosureJaH"'*""^ * mrnnary_ar|gry bypassgrafSJEofTjICardiothorac. SurgT 34,3BO-364Everts, PA et al. (2008) Exogenous application of platelet-leukogel during open subacromial decompression contributes to improvedpatient outcome. A prospective randomized double-blind study. Eury

,_203-21063 Schnabel, LVTeTal. (200/) Platelet nctrplasma (PRPTehhances

anabolic gene expression patterns in flexor digitorum superficiafistendons. J. Orthop. Res. 25, 230-240

64 Mishra, A and Pavelko, T. (2006) Treatment of chronic elbow tendinosiswith buffered platelet-rich plasma. Am. J, Sports Med. 34,1774-1778

65 Bieiecki, T. et al. (2008) Benefit of percutaneous injection of autologousplatelet-leukocyte-rich gel in patients with delayed union andnonunion. Eur, Surg. Res. 40,289-296

167