Cresci sinistra Layout 2 18/02/16 15:17 Pagina 162 ... 3_2015-1.pdf · Alessandro Crisci, Francesco Placido, Michela Crisci, Annamaria Bosco Service of Dermosurgery and Skin Transplantation,

Alessandro Crisci, Francesco Placido, Michela Crisci, Annamaria Bosco

Journal of Plastic Dermatology 2015; 11, 3162

A new instrument aid of plastic surgeon:membranes L-PRF (Platelet-Rich-Fibrin)


Service of Dermosurgery and Skin Transplantation, Nursing Home "Villa Fiorita" Aversa (Ce), Italy.

Introduction

Many results suggest thatplatelets can have a new role in tissuerepair and vascular plasticity, as well asactive elements in immune and inflam-matory responses. They secrete activeproteins and other matters that are able tocondition many processes supportingcells’ intake, growth and morphogenesis.Activated platelets exude and show thesematters. A clot is a natural source ofgrowth factors and cytokines, thanks tosubstances releasing inside it by activatedplatelets, that could be used as therapy tospeed up physiological healing. A lots ofthese stuffs are stocked in α-granules andmay be easily recognize with SEM andimmunofluorescence.Exogenous adding of Platelets RichPlasma (PRP) on wound’s site not onlyspeeds up physiological healing but alsoprovides an additional substrate to tie upfor others cells such as endothelial andsmooth muscle ones, fibroblasts, leuco-cytes, keratinocytes and stem cells as wellas platelets. Among benefits using PRPthere’s safety provided by platelets’ anti-bacterial influence. They actually not onlyrelease substances counteracting bacteri-ums but also take part in bacterial dispos-al during sepsis. Fairly amazing is the recent acknowledge-ment of platelets’ aptitude to reduce pain.Molecular basis have to be study deeper,but an answer could be that plateletsrelease PAR-4 which have antinociceptiveproperties.In 1986 cicatrizant factors derived by

human self platelets (PDWHF) have beenproposed by Knighton et al.1 to help heal-ing of recalcitrating sores and induce for-mation of granulation tissue in early heal-ing’s step.There are any morphological differences ofthick fibers in PRP’s kinds by SEM. Therearen’t many thin fibrin fibers in LPRP(lyophilized PRP) and FPRP (Freshly-madePRP), on the contrary they form a denselayer over thick fibers in HPC (HumanPlatelets Concentrate) (Figure 1).Thin fibers present in HPC could be relat-ed with high initial platelets concentra-tion in HPC (3-5 x 1011 platelets/l),when the local activity in favour of coag-ulation can be improved by amplificationof prothrombotic stimulus, that leads to asuch explosive production of thrombinwith a consequential increase of fibergen-esis on the platelets surface with fibrin’sformation and its polymerization.The HPC also has an higher concentra-tion of fibrinogen (3,5 mg/ml) than FPRPand LPRP and it takes part in secondarynet over tick fibers 2.There are a lots of adhesive proteins on fibrinreticulum: fibrinogen (FG), fibrinonectin(Fn), vitronectin (Vn), thrombospondin-1(TSP-1). Fn takes part to healing woundsand promote mitogen activity PlateletsDerived Growth Factor (PDGF).Among growth factors stored in plateletsand useful wounds' healing there arePDGF (isoform -AB and -C); there are alsoVascular Endothelial Growth Factor(VEGF), Transforming Growth Factor β1

A new instrument aid of plastic surgeon: membranesL-PRF (Platelet-Rich-Fibrin)

The PRF is distinct in P-PRF (Platelet Rich Fibrinpure) and L-PRF (Fibrin Rich in Platelets withleukocytes). In vivo at the height of the forma-tion of the fibrin clot, platelets bind to fibrin β-integrin and the clot shrinks. The PRF clot formsa strong fibrin matrix with a complex three-dimensional architecture. Quantifying PDGF-BB,TGF-1β and IGF-1 in the PPP and the PRF, theanalyzes revealed that the slow fibrin polymer-ization during the processing of PRF leads to theintrinsic constitution from platelets of cytokinesand glycan chains in the meshes of fibrin. Thecentral part of the PRF has massively plateletstrapped in the mesh of fibrin. The localization ofplatelets in the PRF was examined by immunos-taining and Scanning Electron Microscope(SEM). Compared to the membrane PRF tabletdry gauze (G-PRF), the conservation of the levelof the plasma, of the fibrin 3D, and platelets ismore intact in preparations of membrane PRFwith compression system (PRF-C) metal. The dis-tribution of platelets in the membranes C and G-PRF-PRF was analyzed by SEM and immunocy-tochemistry. The fibrin rich platelet pure (P-PRF)and the leukocyte-platelet rich fibrin (PRF-L) arebiomaterials solid fibrin or not containing leuko-cytes. The problem of the concentration ofplatelets does not exist in the PRF, in that all theplatelets of the blood sample taken are activatedand integrated in the matrix of fibrin in the clot.Approximately 97% of platelets, and more than50% of the leukocytes are concentrated in thePRF clot that showed a specific three-dimension-al distribution. Almost all platelets (>97%) wereabsent from tubes of the groups tested afterextraction of the membrane PRF. In the red partof the PRF clot, clots have GR in fibrin network.

KEY WORDS: Growth Factors, Fibrin Rich of Platelets, L-PRF Wound Box.

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Cresci sinistra_Layout 2 18/02/16 15:17 Pagina 162

A new instrument aid of plastic surgeon: membranes L-PRF (Platelet-Rich-Fibrin)

Journal of Plastic Dermatology 2015; 11, 3 163

(TGF-β1), basal Fibroblasts Growth Factor(bFGF) mainly FGF-2; Epidermal GrowthFactor (EGF); Hepatocytes Growth Factor(HGF); Insulin-like Growth Factor (IGF).The members of TGFβ family are veryimportant in wounds healing and forma-tion of scar tissue. TGFβ function is upregulated by a secreting latent form, thatcan down regulate angiosperms even if itpromotes matrix proteins' production.Platelets are a rich source of cytochines andchemokines, owning an important role inwounds healing. An example is RANTES, a chemokine released by a P-Selectine/Plateletsdependent mechanism on inflamed endothe-lium.Platelets are an important source also ofmatrix metalproteases (MMP) (MMP-2,MMP-9, ADAM- 10, ADAM- 17, ADAMTS-13) like also tissutale inibithors ofmetalproteases (TIMP 1-4). The MMPsare stored in α-granules and also in cyto-plasmic membrane's vesicles. The fibrinogen may improve woundscicatrization, increasing both cells’ prolif-eration and migration, it is linked with Fnin fibers regardless of the formation of fib-rin. Fibrin is important in wounds heal-ing, in fact the outcome of healing isinfluenced by the structure of fibrin inwound’s site (thickness of the fibers,number of branch points, porosity andpermeability of the clot) 3 (Figure 2).If a wound doesn’t heal in a quick andspecific sequence, or if the healingprocess doesn’t result in a structuralintegrity, the wound will be consideredchronic. Chronic wound’s healing is simi-lar to the process of acute one, but granu-lation tissue will be made, often withexcessive fibrosis that leads to scar con-traction and loss of function.Chronic wounds and their treatment arean adequate burden on the health systemand this will continue to be a therapeuticchallenge. Because of the heterogeneity ofpatients, multiple etiologies and lack ofanimal models, the research in this area isdifficult and complex.Despite this problems, various pathogen-ic local factor, cellular and molecular,have been identified. Medical therapy isstill the standard choice in ulcers’ treat-ment. If it fails, a surgery treatment is nec-

essary. Skin grafting with partial depth isessential in chronic ulcers’ treatment andis often used to cover or seal not healedextended ulcers. Before the transplant, ulcer is debrided:skin is lipodermatosclerotic, tendonsexposed and subcutaneous calcificationsare removed to create a vascularizedwound, suitable site for a skin cut meshto make intimate contact with the site ofthe ulcer and facilitate the flow of blood.If the formation of a chronic ulcer createsproblems, it will be possible to create anacute injury, because the morbidity back-ground that first created the ulcer is stillpresent.It is clear that the fibrin clot rich inplatelets constitute a bioactive reservoir.An high hematocrit or low level ofplatelets can be a limiting factor and moreresearch are necessary to establish theexact number of platelets for their appli-cation. In addition to secrete proteins,platelets release diffusible stuffs of lowmolecular weight and large amounts ofmicro particles carrying proteins such asTF or IL-1 which are prothrombotic sub-stances. So it could be not recommendedthe use in patients with thrombotichereditary risk factors. The concomitantuse of antiplatelet drugs could theoreti-cally limit the effectiveness.Aspirin reduces platelet secretion andshould therefore be avoided in the daysprior to the preparation of autologousPRP, because it inhibits COX enzyme.After first massive release of growth fac-tors, platelets synthetize and release newones for the rest of their lives (7-10 days).The PRF® (Fibrin Rich of Platelets) it’s anew generation of platelets concentrate,obtained by centrifugation of autologousblood, without adding biological sub-stances. It contains a polymer matrix offibrin, leucocytes, cytokines and stemcells. It is distinguished in P-PRF (PureFibrin Rich in Platelets) and L-PRF(Fibrin Rich in Platelets with Leucocytes). The PRP have a transient effect onwounds healing and also bovine throm-bin increases the risk of coagulopathy,that does not happen with the PRF. It hap-pens a natural clotting process that allowsan easy collection of leucocytes and PRF

in clot. Fibrin gels are desirable as scaf-folds in tissue engineering for many rea-sons. The main one is related with thecompatibility with cell’s life of fibrin,which is different in many componentsand manufacturing processes of scaffolds.Fibrin is a natural substance producedcompletely biodegradable, which facili-tates the transition to a new extracellularmatrix. In vivo at the peak of clottingplatelets bind fibrin and β-integrin andthe clot shrinks. It shrinks also againstedge of wound’s site creating tensionswhich direct new temporary matrix. PRFclot is produced by a natural polymeriza-tion process during centrifugation, andits architecture of natural fibrin may beresponsible of slow release of growth fac-tors and matrix’s glycoproteins.This slow release is unthinkable in mostof PRP techniques because of platelets’sudden activation.PRF clots are directly used to fill a cavityduring a lipostructure in plastic surgery.Even if platelets growth factors have animportant role in PRF biology, the archi-tecture of fibrin and leucocytes’ contentare two data keys.Platelets and leukocytes distributioninside fibrin’s clot was evidenced byblood counts, photonic microscopy andSEM.A good approach for PRF preparationmust separate platelets and erythrocytesand concentrate them without any dam-age or lysing platelets, of course. Growthfactors stored inside α-granules are notactive during secretion, they fuse withmembrane activating themselves.So if platelets are damaged during pro-duction of PRF they won’t produce bioac-tive growth factors. They are very unsta-ble and sensible to all stressing issuesduring processing and application phas-es; according to this reason also growthfactors’ concentration could be influ-enced by manipulation during blood pro-cessing.So it is important also the type of cen-trifugation, it must have specific features,such as Initial Start Low, high rpm inmiddle phase and Final Stop low 4.It also have to be done at specific temper-ature and in a strict time




Material and methods

The protocol of PRF’s prepara-tion it’s very easy: blood have to be cen-trifuged within 2 minutes following thisprogram: 30” of acceleration; 2’ at 2700rpm; 4’ at 2400 rpm, 3’ at 3000 rpm; and36” of deceleration and stop. PRF’s clotshave to be collected and RBC are removedwith scissors without any PRF’s damage atmacro level (processing protocol, Nice,France). The resulting product it is madeby three levels: PPP (Plasma Private ofPlatelets at the top) PRF (clot in the mid-dle), RBC (in the bottom) (Figure 3).Fibrinogen is concentrated in the middleand top of test tube at first, between RBCin the bottom and acellular plasma at thetop. PRF’s clot make a strong fibrin matrixwith a complex tridimensional architec-ture (Figures 2, 3, 7), in which are con-centrated the most of platelets and leuko-cytes. The compression of the clot withdry gauze (G-PRF) causes a reduction ofisoforms of PDGF in comparison with C-PRF (PRF membrane made with a com-pression system) (PRF box) that stimu-lates in a more efficient way the cellularproliferation and neovascularization 5.Quantifying PDGF-BB, TGF-β1 and IGF-1 in PPP and PRF, analysis revealed thatslow polymerization of fibrin duringmanufacture of PRF leads to secretion ofcytokines and glican chains by plateletsinside fibrin’s mesh. Analyzing threeproinflammatory cytokines (IL-1β, IL-6,TNF-α), an inflammatory cytokine (IL-4)and angiogenesis promoter (VEGF), ithave been show that PRF could be a cruxin immune modulation with skills ininflammation control. PRF, in spite ofother platelets concentrates, could be ableto release progressively cytokines duringremodeling of fibrin’s matrix.Many studies have proved that the L-PRPhave antimicrobial effects, but withoutundesired inflammatory reactions. ThePRF allows to surgeon to provide directlya natural healing response and can stimu-late the formation of vital blood vessels,adipocytes, collagen deposition thatseems to resist over time also withoutwound, of course 6.Platelets’ cytokines and leukocytes have

an important role in this biomaterial, butfibrin’s matrix and determinants areresponsible of real therapeutic enhance-ment of PRF. Cytokines are immediatelyutilized in wounds healing.A fibrin glue, enriched with cytokine(such as PRP) with a great uncontrollableeffect have a short duration, it is better aphysiological matrix of fibrin (like PRF),with better effects 7.PRF’s advantages over PRP are:1- None biochemical blood manipulation;2- Simplified and cheaper manufacture;3- Use of bovine thrombin and anticoa-

gulants are not requested;4- Positive healing thanks to a slow poly-

merization:5- More efficient cellular migration and

proliferation;6- PRF have a good effect on immune sys-

tem;7- PRF helps haemostasis.

The mechanism involved in PRF’s forma-tion it’s fibrinogen concentrated at the topof tube test that combines itself with cir-culating thrombin produced by centrifu-gation to make fibrin. The centre of PRFshows many trapped platelets in fibrin’smeshes. The success of this techniquedepends entirely by time elapsed betweenblood collection and its centrifugationthat have to be done in less time as possi-ble, also by manufacturing temperatureand type of tube test. Blood samples haveto be collected from patients with noassumption of aspirin or others anticoag-ulant drugs 2 weeks before. Dohan et al.(1988) revealed a slower release ofgrowth factors and observed better heal-ing skills in PRF than PRP. It have beenproved also that cells are able to migratein fibrin’s mesh. The slow polymerizationmode give to PRF’s membrane a physio-logical architecture particularly favorableto support healing process. Platelets local-ization in PRF have been examined byImmune coloration and SEM. In previousstudies clot’s compression to make a PRFmembrane have been done with a humidor dry gauze. However they aren’t worried about thiscompression, probably platelets damagesand loss of Growth factors.

Su and Burnouf showed that high quantitiesof growth factors are removed by compres-sion. So compression process could influ-ence clinical efficacy and quality of PRF’scompounds as graft material.The levels of growth factors after differenttypes of compression have been valuedwith biological dosages and cytokine-anti-body techniques. Among PRF’s membranecompressed with dry gauze (G-PRF), theconservation of plasma level, fibrin 3Dmesh, and platelets it’s more undamaged inPRF’s membrane compounds with metalcompression system (C-PRF).The humid weight of PRF’s membranedecreased from 2.18 g to 0.35 g withmetal compression and 0.04 g with gauzecompression (decrease of 98% vs 84%).Among tested growth factors, the PDGFcontained in C-PRF it’s more and stimu-lates, significantly cell proliferation andneovascularization. The C-PRF could beuseful for grafting, reducing loss of bioac-tive factors. An important skill of PRF isthat resulting fibrin gel it’s more stiff thanPRF with adding of thrombin (PRP).Fibers density and branch points densityof fibrin’s mesh mainly regulate stiffnessof fibrin’s gel and these parameters arerelated with quantity of thrombin indose-dependent mode (Figure 2).It’s necessary so establish a standard pro-tocol to manufacturing PRF that satisfiedthese rules:1- Platelets growth factors have to be pre-

served to stimulate surroundingpatient cells;

2- Platelets have to be collected in fibrin’smesh with minimal damage or activa-tion;

3- Fibrin’s 3D mesh have to be used asscaffold for nearby patient cells.

The membrane samples PRF were exam-ined by SEM and with the immunocyto-chemical method by Kobayashi et al. 2012 5.The C-PRF was divided into 3 regions ofequal length and the presence of plateletsin each region is been observed at S.E.M. The region 1 is the closest to the red clotand presents numerous platelets aggre-gated and there are some lymphocytesand other white blood cells. The numberof platelets decreases with increasing dis-




The fibrin gel instead have a regulararrangement of pores and reticular fiberswith short and thin and not totally acellu-late and at SEM have been observedplatelets and leukocytes (Figure 5).In studies performed on horse by Textor 8

(2014) and Mc Callan 13 (2014) the FibrinGel containing Platelets, (PRFG) havelarge dense fibers and randomly arranged(with average diameter of 117.7 ± 10.53nm). The fibers of fibrin gel are smaller(56.8 ± 5.11 nm). The incorporation ofplatelets in fibrin gel thus resulting instructural alterations and an increase ofthe concentration of growth factors andcan ultimately improve the performanceof the insertion of cells in the scaffoldafter the transplant.By virtue of the content of growth factors,platelets directly contribute to thegrowth, development and restoration oftissues. The PDGF and TGF-β1 are themost abundant growth factors containedwithin the alpha-granules of platelets andare released into the extracellular spaceafter platelet activation. These cellularfactors direct the proliferation, cell differ-entiation, matrix production, angiogene-sis and wound contraction, supplementa-tion of growth factors improves the sur-vival and differentiation of cells trans-planted into a number of materials andtreated tissues (Figures 6, 7). The addition of platelets to fibrin gels:(PRFG) implies an increase of the diameterof the fibers and the porous decreasing ofthe areas and increasing the rigidity of thefibrin gel. The measured diameter of thefibers has been associated with a lower con-centration of platelets (100 x 103 plate-lets/µl) that approximates the systemic con-centration of platelets in normal horses.The explanation may be that less tensionis applied to the fibers by the lower num-ber of platelets. The porous areas and thepercentage of porosity are importantstructural indices of any biological scaf-fold. The larger pores favor the innergrowth and cell proliferation while thesmallest pores promote cell adhesion dueto a greater surface area. The concentra-tion of leukocytes present in PRP and inPRFG is controversial. However, the spe-cific effect of the concentration of leuko-

tance from the clot red. In region 2 (cen-ter) present fibrin fibers and someplatelets. In region 3 the reticle of fibrin isvery obvious, while platelets are few(Figure 3).The distribution of platelets in the mem-branes C-PRF and G-PRF of the region 1was analyzed by SEM and with theimmunocytochemical.In the C-PRF the reticle of fibrin is com-pletely covered by many aggregatesplatelets of and lymphocytes (Figure 4),whereas in the G-PRF the reticle of fibrinis fully pressed in a film form and can beobserved few platelets. These results wereverified by the detection. of positive cellsto the antibody anti-CD41 throughimmunocytochemistry, in fact in the C-PRF on one side of membrane are accu-mulated numerous platelets CD41-posi-tive and some platelets are located in themembrane.On the opposite side of this there are fewplatelets. In the G-PRF instead to theplatelets aggregates were not found onboth membranes of PRF but only in itwith a higher density than that of C-PRF.In comparing the growth factors con-tained in C-PRF and G-PRF the first con-tains a higher concentration of TGF-β,PDGF-AB, PDGF-BB, EGF, FGF-4, IGF-IIand VEGF-D.The discovering of the study of Kobayashiet al. 5 is that the platelets are not equallydistributed in and on the surface of theclot PRF, although it was considered to bea gel with a uniform concentration ofplatelets. Therefore in a clinical conditionin which factors growth providedplatelets are expected and desired, shouldbe used an adjacent region to the redthrombus which is richer rich in platelets.Based on the concept that the serumretained in the PRF clot may contain highlevels of growth factors released fromplatelets that are more or less active dur-ing centrifugation, it wasn’t attempted tosqueeze all the plasma with full compres-sion of the PRF clots.The tendency to a higher level: growthfactors in the C-PRF compared to G-PRFcan be attributed to the growth factors(FG) Platelet-derived (PDGF-AA, PDGF-AB, PDGF-BB). This result may be due to

fibrin because the reticle of fibrina canabsorb directly the FG or could trapserum albumin or the heparin and thusindirectly to keep the FG.It's almost impossible to count and adjustthe number of platelets in the preparationsof PRF before clinical use. Therefore, theclinically most effective way to control thequality of the results is to use the closestregion to the PRF clot GR.

Results

The L-PRF in SurgeryThe fibrin is a useful substrate for purpos-es of bioengineering and is one of themost popular hydrogel in the field of tis-sue engineering and regenerative medi-cine. The transplanted cells require spe-cific signals from the extracellular matrixto survive. Anoikis is a term used todescribe the premature death of cells thatdo not receive these signals mechanicaland chemical from the matrix, and thisphenomenon is considered a major causeof transplant cell failure. The fibrin rich of platelets pure (P-PRF)and the Leukocytes-Fibrin are Rich ofPlatelets (PRF-L) are biomaterials solid fib-rin or not containing leukocytes. In thesetechniques the platelet activation is part ofthe process of production and can be nat-ural (L-PRF) or artificial (P-PRF), but italways occurs during centrifugation andleads to a strong final architecture of fibrin.The L-PFR is a preparation with leuko-cytes and with a high density of the fibringrid. These products exist in the form ofactivated gel and cannot be injected orused as traditional fibrin glue. However,because of their strong fibrin matrix canbe treated as a solid material for applica-tions that have been proposed, with inter-esting results in general surgery, but theseapplications are still in the experimentalstage as they require to find a way to useclots in each specific surgical procedure.Fibrin create a provisional matrix in thespace of the transplant, but its fibers donot have directionality and tension, it hasfew growth factors associated content.




cytes on the formation of clot, on the for-mation of fibrin gel or on the rigidity ofthe clot has yet to be studied. The PRPused for the formation of PRFG has a con-centration of WBC intermediate (average:9.34 x 103 leukocytes/ml) (range: 3.2 x103 to 16.0 x 103 leukocytes/ml) 8.From a clinical point of view, L-PRF hasexcellent handling properties: the indi-vidual clots L-PRF are transformed intomembranes of appropriate size and thick-ness thanks to the new "L-PRF WoundBox"; more membranes can be joinedtogether and will serve to create a mem-brane bioactive larger to cover and formlarge insert. The membrane of L-PRF canbe cut to measure. Being flexible enoughadapts well to different anatomical areas.From a legal point of view, a doctor beingauthorized to intravenously needle-stickis also authorized for blood sample.Moreover, by EU regulation, then withthe law valid throughout Europe the L-PRF having no addition of substances isnot an emo-derived but is included in thecase of re-engagement of autologous cells.As the healing that occurs with this tech-nique is neither first nor second inten-tion, it is called "healing by second intentionmodified" 9. The family L-PRF fits theneeds of the surgeries. As clots and themembranes L-PRF has a shape and a vol-ume easy to combine with the majority ofsurgical techniques, such as filling andinterposition of biomaterials of healing oras protection membranes for the healingof wounds. These membranes are alsostrong and provide a slow release of sev-eral growth factors for long periods.Finally, it is easy to prepare in large quan-tities and inexpensive, which makes itparticularly suitable for daily clinicalpractice. It is used in particular in thetreatment of skin ulcer.

The L-PRF in VitroIn vitro behavior of a membrane and L-PRF and Gel P-PRP (Platelet Rich Plasmapure) (PRFG-endorest) were comparedthrough the evaluation of the slow releaseof growth factors and matrix molecules.These two families of gel were placed in aculture medium for 7 days, and the ver-

sions slowed of 3 factors of key growth.(TGF-β1, PDGF-AB, VEGF) and 3 coagu-lation proteins and matrix (TSP-1 [throm-bospondin-1]) (Fn) (vitronectin) werequantified experimentally seven times (at20', 1h, 4h, 24h, 72h, 120h, 168h)(Figures 9, 13) 10.These studies revealed that the productshave two very different profiles: the mem-brane L- PRF remained solid and intactafter 7 days and continuously releases alarge amount of growth factors, a signifi-cant part of it is produced by cells insideof the membrane.In contrast the gel P-PRP clarified releasesmost of the growth factors in the earlyhours and is fully dissolved after 3 days(Figures 10, 13).The leukocytes present in the L-PRF arenot only inflammatory cells, as they alsohave anti-nociceptive effects through sev-eral chemokines, anti-inflammatorycytokines (IL-4, IL-10, IL-13) and opioidpeptides (β-endorfine- dimorfina-A etc.)and therefore can promote a clinically rel-evant inhibition of pathological pain (thecentrifugation process. can turn gentlystimulate pathologically inflammatorystate or destroy leukocytes). Many typesof cells are present in these preparations.The leukocyte formula is an importantparameter: the lymphocyte populationsare very different. does not have quite thesame impact of monocytes and granulo-cytes. Also, many other cells, such as cir-culating stem, can be found in a plateletconcentrate and not be neglected.Was observed a certain amount of GF inthe serum released by PRF (PRFR) and inthe serum supernatant (SS) immediatelyafter the formation of the PRF. It is afound an additional release from PRF GFup to 300' (5h). The content of GF in SSis ≅ 7 ng/mL (PDGF-AB), 9.5 ng/mL(TGF-β1), 0.1 ng/mL (VEGF) and consti-tutes a good indication of the basal levelof GF in PRFR trapped in the clot. Thereconcentration of GF issued by the PRF issignificantly lower than that found inplatelet lysates and is lower than the con-centration present in the whole bloodused to produce PRF. The PRF and the SScontain large amounts of GF and mustnot be discarded as they can be useful in

the treatment of the patient 11.The inflammatory liquid in the PFR (richin growth factors and serum protein) iscollected in the container and, PRF mem-branes are kept in a humid environmentof serum. This is an effective method froma biological point of view. A new devicetested by us for the preparation and stan-dardization of L-PRF clots and mem-branes is the L-PRF Wound Box.The clot L-PRF contains almost all theplatelets and more than 50% of the whiteblood cells initially collected, also has astrong architecture of fibrin and a specialthree-dimensional distribution ofplatelets and leukocytes. One solution isto keep the clot in a metal container andpress them in sterile membranes with asterile plate metal when necessary.This device allows the preservation of theclot in a moist and sterile for 1h andallows an increased release of growth fac-tors · total. The L-PRF Wound Box® is aversatile tool where PRF clots can betransformed into membranes. The aver-age quantities of produced PDGF-AB aresignificantly higher in each experimentaltime and the TGF-β1 (Figure 10) andVEGF (Figure 11) are significantly higherduring the first 4h. The explanation ofthis result is quite simple: by using thePRF Box, the compression process of theclots in the membrane is performed by aslight compression, slow and homoge-neous, and the final membrane alwaysremains homogeneously wet and soakedin serum. This gentle method avoids the extractionand the loss of a significant amount ofgrowth factors, and is particularly evidentfor the PDGF-AB, because this growthfactor is released only by the platelets. Onthe contrary, will not influence otherintrinsic factors of growth that arereleased slowly in high quantities for sev-eral days. The amount of released VEGFand TGF-β1 are produced by leukocytesmassively.A process for the collection of blood andpreparation is not standardized, slow andinadequate, leads to a small mass of fibrinPRF-like, with the polymerization of fib-rin unstable (resulting in weaker mechan-ical properties) and a growth factor




unknown and irreproducible. Also it isvery difficult to separate these small mass-es of fibrin at the base of the GR, resultingin a heavy load of red blood cells in theproduct 12.

DISCUSSION

1- Being the PRF a product belonging tothe same body; the availability of thisbiomaterial in greater quantities is dif-ficult. Therefore, its use in surgicalprocedures must be well planned.

2- The PRF has circulating immune cellsand antigenic molecules that preventits use as allogenic material and thereis a increased risk of transmission ofinfectious agents.

Platelets, fibrin and leukocytes naturallyact in synergy to promote wound healingand regeneration tissue and the conceptof platelet concentrates for surgical use isto multiply this effect coagulation/regen-eration on a surgical site or wound.According to the classification POSEIDOall products in this category are groupedunder the general term of platelet concen-trates (HPC), whatever their form or con-tent cellular, also it is important to high-light the key influence of leukocytes andthe structure fibrin in the clinical poten-tial or experimental effects of these prod-ucts and that each product refers to a spe-cific biological fingerprint.At this point of our knowledge, amongthe parameters kept outside of this classi-fication system we have: the concentra-tion of platelets, the concentration ofleukocytes and. proportions of varioustypes of leukocytes. The problem of theconcentration of platelets does not existin PRF, in that all the platelets of the bloodsample taken are activated and integratedin the matrix of fibrin in the clot.Regarding the concentration of leukocytesand their formula, their influence has yetto be studied carefully, since their presenceor absence may explain the contradictoryresults that are observed (Tables I, II).The second generation of PRF, one withleukocytes (L-PRF) recently developed, is

expressed as a three-dimensional bioma-terial handle, which does not melt, but isdestroyed by remodeling over time, simi-lar to the natural blood clot.There were no statistically significant dif-ferences between the concentrations base-line WBC of 7.4 x 103/ml and platelets.166 x 103/ml. There are correlationsbetween blood levels and basal concen-trations of FG. There is a significant corre-lation between the number of platelets andrelease of TGF-β1 (p = 0.005) and PDGF-BB (P = 0.04). The TGF-Β1 is quantitative-ly greater in group PRF slow-release com-pared to immediate-release (Figure 6).It was reported that WBC trapped in PRFmatrix represents the main source of thisadditional release of TGF-β1, and WBCcontinue to produce this FG clot withinthe PRF for several days (about 7).The PDGF-BB is almost completely con-tained in the alpha-granules of plateletsand is released upon activation, which iswhy its production is highest in the PRPand PRF activated and reduces lensshapes. It is possible that the haematolog-ical values can not be used to predict theimmediate or slow release of growth fac-tors in the PRF 13.In the study of Dohan Ehrenfest et al. 14,we wanted to determine the cellular com-position and organization of this biomate-rial three-dimensional autologous andusefulness of different tubes (Dry glass,coated glass, plastic) but all without gel.Approximately 97% of platelets, andmore than 50% of the leukocytes are con-centrated in the PRF clot that showed aspecific three-dimensional distribution.The platelets and fibrin present in largequantities in the first millimeters of themembrane beyond the limit of the redblood cells. There is the presence of dif-ferences in the architecture of PRF usingvarious types of tubes.

Analysis of platelets and leukocytesAlmost all platelets (> 97%) were absentfrom tubes of the groups tested after 'ex-traction membrane PRF. In the groups test-ed, the level of leukocytes decreased signif-icantly compared to the control group (p <0.01) more than half of leukocytesappeared to be disappeared (Table 1).

Platelets and leukocytes missing aretrapped in the PRF matrix when using themethod of collection with scissors.The absence of a difference between thetwo groups tested (p > 0.05) seems toindicate that the brutal compression ofthe clot does not affect the possiblerelease of cell bodies trapped within thefibrin matrix.In the groups tested, the concentration oflymphocytes is significantly lower, whilethat of neutrophils was significantly higher(p < 0.01) in the control group (Table 2).This would indicate that the lymphocyteswere more than other leukocytes trappedin the matrix of PRF. Finally, the meanplatelet volume (MPV) is reduced signifi-cantly between the groups tested andcontrol groups (p < 0.01), it decreasefrom 9 µm3 (range: 8-11 µm3) in wholeblood to 4.7 µm3 (range: 4.5 to 5.8 µm3)in the groups tested. This phenomenoncould be due to increased plasma osmo-larity in the tubes after the activation ofthe coagulation cascade.

Study of Optical MicroscopyHistomorphometric analysis was per-formed using an optical microscope witha total magnification of 100 x. An eye-piece 10 mm with a pattern with 100divisions was used to measure the per-centage of coverage of the total lengthwith at least one cell layer in each section.With hemalaun-eosin staining the fibrinmatrix is homogeneous in pink, while theaggregated platelets are dark blue/purple(Figures 12a, b). The GR and cytoplasmof leukocytes are dark pink and not easi-ly detectable. The nucleus of the leuko-cytes are colored in blue with hemalumenand are not easily distinguishable fromplatelet aggregates. With the tri-colorMasson (modified from Godman) the aggre-gated platelets are still dark blue, but theGR are easily identifiable because coloredred. Leukocytes are still difficult to high-light within platelet aggregates, however,the line of demarcation between GR andplatelet aggregates/leukocytes is very clear(Figure 12c, d). The line of distinctionbetween GR, leukocytes and plateletaggregates is not obvious.




yellow/red more platelets (and leukocytes)are grouped into the veins of platelet con-centrations central or centrifuges. Theseveins have a high density of platelet/leuko-cyte to the interior of a matrix free of cells.This structure is similar in all coagulates,regardless of the patients, the type of testtubes and the compression method.The platelet count showed clearly thatthere are almost no platelets let layer GR,the PPP and the inflammatory liquid aftercompression of the PRF clot. So most ofthe platelets from the whole blood sampleis collected in the PRF membranes.The white blood cell count has confirmedthat more than half of leukocytes istrapped in the PRF membranes and Thesmall lymphocytes seem attracted soselected as confirmed by SEM (Tables 1,2). These leukocytes do not appear to bedamaged during the preparation of PRF.This result has a strong clinical impact asthe amount of leukocytes placed at theinside of the membranes is considerableand small lymphocytes are particularlyefficient in the regulation of inflammatory

Rating at SEMObserving the PRF clot at SEM at lowmagnification (x15) showed that the clothas a central concavity which is a resultnot real fixation. In the red part of thePRF clot, I have coagulates GR in fibrinnetwork (Figure 13). The GR are normalbut the fibrin grid appears immature.The boundary between the red and theyellow of the clot (area buffy coat), theSEM examination showed leukocyteswhich appear as spherical structures withirregular surface (Figure 13 a).The majority of these is small (6-8 µm indiameter) and thus may be mainly lym-phocytes. Platelet aggregates appear alongthe fibrin filaments (Figure 4 b).Beyond the area buffy coat are distin-guished two different areas: the first con-sists of thick filament of fibrin and a fewscattered GR probably from contamination;fibrin appears to be mature (Figure 14); thesecond consists of that vein of plateletsthickened observed under the opticalmicroscope, characterized by plateletsand fibrin in dense clusters and large due

to extensive aggregation and coagulation(Figure 15). This aggregate is constitutedby a thick grid and solid and plateletsappear to significantly activated duringthe preparation protocol of PRF.A higher magnification fibrin is clearlyorganized in parallel fibers very thick anddense. In it is impossible to distinguishthe cellular elements content.

CONCLUSIONS

The highest density of plateletsand leukocytes was found in the first mil-limeter of the clot to limit yellow with thered. The delivery of platelets and leuko-cytes become lower as we move to theend of the clot and have not been foundmore platelets and leukocytes over thefirst half of the clot yellow.In the first 2 mm over the edge betweenthe red and the yellow clot distribution ofplatelets and leukocytes is fairly homoge-neous on hall the length of the clot.As you move from the edge of the

Table 1. Leukocytes, RBC and Platelets number in whole blood (control group) and red clot after PRF membrane collecting (test group) (by Dohan Ehrenfest et al. 2010 modified).

Leukocytes/µl EtiologyRBC/µl Platelets/µl

Mean Range Mean Range Mean Range

Controll 6.900 6.100-7.800 5.19 (106) 5.01-5.52 (106) 2.66 (105) 2.18-3.09 (105)

Serie 1 3.500 3.000-3.800 5.89 (106) 5.75-6.08 (106) 6.000 4.000-8000

Serie 2 3.600 3.300-4.000 5.84 (106) 5.78-5.91 (106) 7.000 6.000-9000

Table 2. Leukocyte formula stabilized in whole blood (control group)and red clot after PRF membrane collecting (test group) (by Dohan Ehrenfest et al. 2010 modified).

Tipo di cellula

Whole blood (%) Serie 1 (%) Serie 2 (%)

Mean Range Mean Range Mean Range

Neutrophils 51.8 49.7-53.2 72.1 66.1-77.1 66.4 60.9-71.4

Eosinophils 2.9 2.3-3.1 6.1 3.4-8.8 5.1 3.9-6.1

Basophils 0.5 0.3-0.8 0.1 0.0-0.3 0.4 0.1-0.9

Linfocytes 37.7 35.1-39.2 17.5 15.0-20.4 24.8 21.4-28.0

Monocytes 71.1 6.8-7.6 4.2 1.1-7.6 3.3 2.5-5.0

Total (Mean) /µl 6.900 (100%) 3.500 (100%) 3.600 (100%)




reactions. Furthermore, the cellular com-position of the L-PRF implies that thisbiomaterial must be handled with care toensure that the cell contents will be keptalive and stable. The microscope has shown that the pho-tonic platelet distribution and leukocytesin the clot is not uniform. Platelets and leukocytes are concentratedin the layer which is located between theclot and the clot red fibrin and arearranged forming a coating on the surfaceof the macroscopic clot PFR. Therefore,the most useful part by a surgical point ofview is the whitish layer intermediate. Soit is necessary to preserve a small layer ofGR at the end of the clot PFR which con-tains most of leukocytes and platelets.The procedure can be done with scissors

and is operator-dependent and thisrequires accurate knowledge of the struc-ture of the PRF. The slight compression of the fibrinmatrix determines that the fibrin fila-ments are condensed and stick together.When the membranes are used in surgeryPRF their reabsorption is slow and facili-tates the remodeling of the fibrin matrixin a scar tissue. For the standardization of the preparationPRF as graft material for tissue regenera-tion, we propose the use of the region ofthe membranes of PRF with the maxi-mum enrichment platelet and, in addi-tion, not to squeeze all the content in theplasma clot PRF. So it is advisable to com-press the clot with a compression device(L-PRF Wound Box). therefore it is dif-

fcult to control accurately the quality ofhuman-derived materials, such as prepa-rations PRF, but it is very important tomake the best quality control on PRF pre-pared before their clinical application.Further clinical studies, histological andstatistical are required to understand thebenefits of this new platelet concentra-tion. However, one can not ignore thatthe one obtained from a sample of autol-ogous blood, the PRF product is scarceand only a limited volume can be used.This limits the systematic use of PRF inGeneral Surgery. Although the potentialapplications of PRF are large, you need athorough knowledge of the operation ofthe biomaterial, its biology, and efficiencyadvocate, limits, to optimize its use indaily clinical practice 15.

Figure 1.SEM images showing a) Platelets aggregate in fibrin clotfrom Human Platelets Concentrate (HPC) (magnificent10000x) and fibrin fibers. B label shows minor smooth fibersthat constitute a secondary mesh on fibrin main fibers.

Figure 2.Difference between PRP (a) and PRF (b) technique in fibrinmolecular architecture. Look at the elastic differencebetween the structures.

Figure 3.The 3 areas of C-PRF and SEM’s observations on mem-brane surface. (A) C-PRF has been divided into 3 regions:Region 1 closest to red clot (RBC), region 2 the middle oneand region 3 that’s the more far by red clot. Platelets local-ization has been observed in region 1 (A), region 2 (B) andregion 3 (C). The concentration of platelets is higher inregion 1 than 3. (modified from Kobayashi et al. 2012).




Figure 4.A) on the edge between red zone and yellow clot by SEM.RBC and leukocytes group appear with spherical structureand irregular surface (white circles). The greatest part ofthem seems to be quite small (between 6 and 8 µm ofdiameter) and, so, could be lymphocytes. B) Platelets wasoften mixed in fibrin mesh, but sometimes, appear likeaggregates (white circles) that have been easily identified.(Magnifier A) 1500x, B) 3500x.(modified from Dohan Ehrenfest et al. 2010).

Figure 5 a, b.PRF clot at SEM. The arrow shows thrombocytes a) board=10 µm b) board=1 µm.

Figure 6.TGF-β1 levels in horse (modified from McLellan, Plevin, 2014).

Figure 7.PDGF-BB levels in horse (modified from McLellan, Plevin, 2014).

Figure 8.Growth factors levels by hours post blood draw.

Figure 9.TSP-1 and fibronectin variations by time. L-PRF versus PRGF(Plasma rich in Growth Factors=P-PRP) (modified from Dohan Ehrenfest et al. 2012).




Figure 10.TGF-β1/PDGF-AB and vibronectin by time. L-PRF versusPRGF (modified from Dohan Ehrenfest et al. 2012).

Figure 11.VEGF variations by time. L-PRF versus PRGF (modified from Dohan Ehrenfest et al. 2012).

Figure 12.Analysis by Optical Microscopy of PRF clots. A e B) Hemalaun-eosin is not sufficient to distinguish vari-ous cell types trapped into fibrin matrix. C e D) Using tri-color Masson coloration it’s easy to distinguish plateletsaggregates by leukocytes (dark blue and RBC (red). Magnification (G) (modified from Dohan Ehrenfest et al. 2010).

Figure 13.A e B) red zone of PRF’s clot. (SEM) contains many RBCtrapped into an immature and very melted fibrin matrix(Magnification A x 750, B x 2000) (modified from Dohan Ehrenfest et al. 2010).




References1. Knighton DR, Fiegel VD, Austin LL, et al.

Classification and treatment of chronic nonhealingwounds, successful treatment with autologus platelet-derived wound healind factors (PDWHF), Ann Surg,1986; 204,322-29.

2. Pretorius E, et al. Ultrastructural comparison of themorphology of three different platelet and fibrin fiberpreparations, The Anatomical Record, 2007; 290,188-98.

3. Del Corso M, Choukroun J, Simonpieri A, et al.Accelerazione dei processi di cicatrizzazione tissutale conun nuovo biomateriale: la fibrina ricca di piastrine(PRF), Odontoiatria, 2007; 4,361-66.

4. Nurden AT, Nurden P, Sanchez M, et al. Platelets andwound healing, Frontiers in Bioscience, 2008; 13,3525-48.

5. Kobayashi M, Kawase T, Horimizu M, et al. A proposedprotocol for the standardized preparation of PRF mem-branes for clinical use, Biologicals, 2012; 40,323-29.

6. Zhao QM, Ding YJ, Si T. Platelet-rich fibrin in plastic sur-gery, OA Evidence-based Medicine, 2013; 1(1):3.

7. Naik B, Karunakar P, Jayadev M, et al. Role ofplatelet rich fibrin in wound healing: a critical review, JConserv Dent, 2013; 16,284-93.

8. Textor JA, Murphy KC, Leach K, Tablin F. Ultrastructureand growth factors content of equine platelet-rich fibrin gels,A J V R, 2014; vol.75, n4, 392-401.

9. Desai CB, Mahindra UR, Kini YK, Bakshi MK. Useof platelet-rich fibrin over skin wounds: modified sec-ondary intention healing, J Cutan Aesthet Surg, 2013;6,35-37.

10. Dohan Ehrenfest DM, Andia I, Zumstein MA,et al.Classification of platelet concentrates (Platelet-rich plas-ma- PRP, Platewlet-rich fibrin - PRF) for topical andinfiltrative use in orthopedic and sports medicine: cur-rent consensus, clinical implications and perspectives, ML T J, 2014; 1,3-9.

11. Su C-Y. How to optimize the preparation of leuko-cyte- and platelet-rich fibrin (L-PRP, Choukroun tech-nique) clots and membranes: introducing the PRF box,

Oral Surg, Oral Med, Oral Path, Oral Rad End, 2010;110,278-80;

12. Dohan Ehrenfest DM, et al. Do the fibrin architectureand leukocyte content influence the growth factor releaseof platelet concentrates? An evidence-based answer com-paring a pure platelet-rich plasma (P-PRP) gel and aleukocyte- and platelet-rich fibrin (L-PRF), CurrentPharmaceutical Biotechnology, 2012; 13,1145-52.

13. McLellan J, Plevin S. Temporal release of growth fac-tors from platelet-rich fibrin (PRF) and platelet-richPlasma (PRP) in the horse: a comparative in vitro analy-sis, Intern J Appl Res Vet Med, 2014; 1,48-57.

14. Dohan Ehrenfest DM, Del Corso M, Diss A, et al.Three-dimensional architecture and cell composition of aChoukroun's platelet-rich fibrin clot and membrane, JPeriodontol, 2010; 81(4),546-55.

15. Chatterjee A, Agarwal P, Subbaiah SK. Platelet richfibrin: an autologus bioactive membrane, APOLLOMedicine, 2014; 11,24-26.

Figure 14.SEM analysis of fibrin yellow clot reveals a dense and maturefibrin matrix with a low quantity of identifiable trapped bodies(RBC, Leukocytes, Platelets aggregates) (Magnificent x 2000)(modified from Dohan Ehrenfest et al. 2010).

Figure 15.A e B) SEM in white veins inside yellow clot, platelets aggre-gates was tightly joined in a dense and mature fibrin matrix.(Magnificent A x 1000, B x 1500)(modified from Dohan Ehrenfest et al. 2010).


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