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www.cellsindentistry.org
Volume 1 Issue 1 January - April 2018
INTERNATIONAL JOURNAL INTERNATIONAL JOURNAL
OF GROWTH FACTORS OF GROWTH FACTORS
AND STEM CELLS IN DENTISTRYAND STEM CELLS IN DENTISTRY
INTERNATIONAL JOURNAL
OF GROWTH FACTORS
AND STEM CELLS IN DENTISTRY
© 2018 International Journal of Growth Factors and Stem Cells in Dentistry | Published by Wolters Kluwer - Medknow
8
Abstract
Original Article
IntroductIon
Inthelastdecades,differenttechniqueshavebeendeveloped
to promote bone and soft‑tissue regeneration.[1] Natural
biomaterials,suchasbovine‑derivedxenogeneicbiomaterials
or allogeneic biomaterials of human origin, must undergo
different purification and processing stages to eliminate
pathogens and minimize the risk of disease and gene
transplantation.[2] Thus, chemical and physical purication
techniques lead to the loss of the regeneration potential of
the biomaterials and reduce their bioactivity.[2]In addition,
synthetic biomaterials are imitatively manufactured and do
not exhibit similarity to the native tissue structure and the
bioactivity of natural biomaterials.[3] Therefore, to identify
Aims:Platelet‑richbrin(PRF)existsinbothsolidanduidforms.Thepresentstudywasthersttoevaluatetheinuenceofhomogeneous
pressureonthegrowthfactor(GF)releaseinpressedPRF‑matricesandplugs.Methods and Material:AsolidPRF‑matrix(208g;8min)
waspressedtoobtainaplug,andapressedPRF‑matrixthatareusedinclinicalapplication.Thereleasedexudateswereevaluatedcomparedto
liquidPRF(60gand3min).TheVEGF,TGF‑ß1andEGFreleasewasquantiedusingELISA.Thebrinstructureandcellularcomponentsin
solidPRFgroupswereevaluatedhistologically.Results:ThepressedPRF‑matrixandPRF‑plugexhibiteddenserbrinstructurecomparedto
thenon‑pressedPRF‑matrix.Onday7,thePRF‑plugandnon‑pressedPRF‑matrixshowedsignicantlyhigherreleaseofVEGF,TGF‑ß1and
EGFcomparedtothatofthepressedPRF‑matrix.TheaccumulatedVEGFconcentrationwassignicantlyhigherinthePRF‑plugcompared
tothatinthePRF‑matrixandnon‑pressedPRF‑matrix.TheaccumulatedEGFandTGF‑ß1concentrationsover10daysshowednostatistically
signicantdifferencesbetweentheevaluatedsolidPRFgroups.TheexudatesreleasedTGF‑ß1andEGFpassively,thatwasonlydetectable
inafter1and7hours.LiquidPRFreleased signicantlyhigherGFsthantheexudatesatallinvestigatedtimepoints.TheearlyVEGFand
EGFrelease inliquidPRF (1 hourto1 day)wassignicantly higherthanthat in thesolidPRF‑matrices. Onday10, signicantlyhigher
accumulatedGFsweredetectedinthesolidPRFgroupscomparedtothoseintheliquidPRF.Thus,thecombinationofbothsolidandliquid
PRFisapotentialtooltogenerateaclinicallyrelevantsystemwithsustainedbioactivity.Conclusions:Theseresultshighlightthepotential
toinuencetheGFsreleaseproleofsolidPRFmatricesbypressureandobtainaclinicallyapplicableplugwithsignicantlyhigherVEGF
release,providingfurtherunderstandingofthereleaseproleofPRFmatricesasadrugdeliverysystem.
Keywords:Growthfactors,liquidplatelet‑richbrin,low‑speedcentrifugationconcept,regeneration,solidplatelet‑richbrin
Address for correspondence: Prof. Shahram Ghanaati,
Department of Oral, Cranio-Maxillofacial and Facial Plastic Surgery,
University Hospital Frankfurt Goethe University,
60590 Frankfurt am Main, Germany.
E-mail: shahram.ghanaati@kgu.de
Access this article online
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DOI:
10.4103/GFSC.GFSC_9_18
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For reprints contact: reprints@medknow.com
How to cite this article: Al‑Maawi S, Herrera‑VizcainoC, Dohle E,
ZrncTA,ParviniP,Schwarz F,et al.Homogeneous pressure inuences
thegrowthfactorreleaseprolesinsolid platelet‑richbrinmatricesand
enhancesvascularendothelialgrowthfactorreleaseinthesolidplatelet‑rich
brinplugs.IntJGrowthFactorsStemCellsDent2018;1:8‑16.
Homogeneous Pressure Influences the Growth Factor Release
Profiles in Solid Platelet‑rich Fibrin Matrices and Enhances
Vascular Endothelial Growth Factor Release In The Solid
Platelet‑rich Fibrin Plugs
Sarah Al‑Maawi, Carlos Herrera‑Vizcaino, Eva Dohle, Tomislav A Zrnc1, Puria Parvini2, Frank Schwarz2, Robert Sader, Joseph Choukroun3, Shahram Ghanaati
Frankfurt Orofacial Regenerative Medicine (FORM) -Lab, Department for Oral, Cranio-Maxillofacial and Facial Plastic Surgery, University Hospital Frankfurt Goethe
University, Frankfurt am Main, 2Department of Oral Surgery, Center for Dentistry and Oral Medicine (Carolinum), Johann Wolfgang Goethe-University Frankfurt am
Main, Germany, 3Private practice, Pain Therapy Center, Nice, France, 1Department of Oral and Maxillofacial Surgery, Medical University of Graz, Auenbruggerplatz 5,
A-8036 Graz, Austria
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018 9
aminimally invasiveautologousregenerationsource, blood
concentrateshave beenintroducedasa promisingclinically
relevantmethod.Bloodconcentrates aregeneratedfromthe
patients’ownperipheralblood,concentratedbycentrifugation,
andcanreleasedifferentgrowthfactors(GFs).[4]
Therst‑generationbloodconcentrates, termedplatelet‑rich
plasma, require multiple centrifugations and the addition
of anticoagulants and external chemical activation
substrates.[5]However,thiselaboratepreparationmayprovide
majordrawbacksforclinicalroutine.Thus,theintroductionof
platelet‑richbrin(PRF)facilitatestheclinicalapplicationof
bloodconcentratesystems.PRFisobtainedfromthepatients’
venous blood by single centrifugation without additional
anticoagulants,makingita100%autologousbloodconcentrate
system.[6]Thefast and easy preparationof PRF makes this
materialclinicallyapplicableandfavorableforclinicians.[7]
During centrifugation, a solid‑brin matrix is generated by
theactivationoftheplateletsthrough their interaction with
the tube surface.[8] The activation of platelets promotes the
release of different GFs.[9] The resultingsolid PRF matrix
consistsofabrinscaffoldthatincludesplatelets,leukocytes,
and plasma proteins. The preparation protocol of the rst
describedsolidPRFrequirestheapplicationofahighrelative
centrifugationforce(RCF).[8]TheroleoftheappliedRCFand
centrifugationtimeintheformation,structure,andbioactivity
ofPRFmatriceshas beenextensivelystudiedbyour group.
Arecent exvivo study reported a protocol modication by
reducingtheappliedRCFfollowingtheso‑calledlow‑speed
centrifugation concept (LSCC), resulting in a more porous
brinmatrixthatcontainedahighernumberofplateletsand
leukocytescomparedtosolidPRFprepared byusingahigh
RCF.[10] Further protocol adjustment to maintain the RCF
andpropose a reduction ofthe centrifugation time ledto a
signicantincreaseinthereleaseofGFs,particularlyvascular
endothelial GF (VEGF) measured in vitro.[11]Moreover,
reducingtheRCFinuencedtheplateletdistributionpattern
throughoutthematrix.ThesolidPRFmatrixpreparedusing
a reduced RCF showed more evenly distributed platelets
comparedtotheaccumulatedplateletsatthebottomofsolid
PRFmatricespreparedusingahighRCF.[11]
InadditiontothesolidPRF,theclinicalneedsforaliquidPRF
requiredthedevelopmentofaliquidPRFwithoutadditional
anticoagulants. Following the LSCC, a liquid PRF rich in
platelets,leukocytes,andGFswasachievedbyasystematical
reductionoftheappliedRCFandcentrifugationtime.[12]The
introductionoftheliquidPRFwidenedtherangeofthePRF
applicationinsurgicalperiodontologicaltherapyandfacilitated
itscombinationwithbiomaterials.
Intheclinicalapplication,solidPRFisprocessedbypressure
to obtain tailored PRF forms that are suitable for specic
indications. For instance, solid PRF matrices are pressed
togenerateplugs that areusedforsocketpreservation after
teethareextracted.[13]Solid‑pressedPRFmatricesobtainedby
pressingthesolidPRFmatrixareappliedinperiodontology
forrootcoverage,incombinationwithbiomaterialsandasa
wounddressing.[14]Duringthepressureprocess,liquidexudate
isreleasedfromthesolidPRFmatrix.Todate,littleisknown
abouttheinuenceofpressureontheGFrelease ofpressed
PRFmatrixandplugandtheirexudates.Toourknowledge,the
presentstudyisthersttoevaluatetheeffectofastandardized
preparationtechniquetogainpressedPRF matrix and plug
comparedtononpressedPRFmatrix.Theaimofthepresent
studywastoanalyzetheeffectofsolidPRFmatrixprocessing
by pressure on the bioactivity, regenerative capacity, and
structureoftheclinicallyusedPRF plugs and pressed PRF
matricescomparedtothenonpressedsolidPRFmatrices.In
addition,thebioactivityofthereleasedexudateswasevaluated
comparedtothatoftheliquidPRF.
MaterIals and Methods
TheapplicationofPRFinthisstudywasinaccordancewiththe
principleofinformedconsentandapprovedbytheresponsible
EthicsCommissionofthestateofHessen,Germany(265/17).
Platelet‑rich fibrin preparation
ForPRFpreparation,sixdonors(threemalesandthreefemales)
were included in the present study.The preparation was
performedaspreviouslydescribed[Figure1].[8]Briey,after
obtaining informed consent from each of the participating
donors,venousbloodwascollectedfromtheperipheralmedian
cubitalvein.SixtubeswereusedtogeneratesolidPRF(Red
tubes [10 ml], Process for PRF™, Nice, France), and one
tube(Orangetube[10ml],ProcessforPRF™,Nice,France)
wasusedtoobtainliquidPRF.Afterbloodcollection,thetubes
wereimmediatelyplacedinpreprogrammedcentrifuges(Duo
centrifuge,ProcessforPRF™,Nice,France)andcentrifuged
accordingtoestablishedprotocols:
• SolidPRFmatrix: 208g 8min
• LiquidPRFmatrix: 60g 3min.
Solid platelet‑rich fibrin processing
Understerileconditions, the tubesweregentlyopened, and
thePRFmatricesweresubsequentlycarefullyretrievedfrom
the tube and isolated from the red blood fraction without
damagingtheinterfaceofthematrix.APRFboxthatfacilitates
homogeneous pressure application was used to maintain
standardizedconditionsduringprocessing thePRFmatrices
toplugsandpressedPRFmatrices.Foreachdonor,onePRF
matrixwastransferredtothePRFbox(PRFbox,Processfor
PRF™, Nice, France), placed into the preformed hole and
carefullycompressedusing the compressing tool according
to the manufacturer’s instructions as reported elsewhere.[15]
Theresultingplugwastransferredtoa6‑wellplate,andthe
releasedexudatewascollectedandtransferredtoa6‑wellplate
usinga pipette. The secondPRF matrix was placed on the
PRFboxgridandcoveredbythepressuretoolandboxcover
withoutactivepressure.After5min,theboxwasuncovered,
andthepressedPRFmatrixwastransferredtoa6‑wellplate.
Theresultingexudatewascollectedandtransferredtoa6‑well
plate.The third PRF matrixwasdirectlyplacedina 6‑well
plateandusedasanonpressedcontrol[Figure2].Thesame
procedurewas repeatedwiththree additionalPRFmatrices,
Figure 1: Flowchart of the study design
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018
10
and the resulting solid PRF plug, pressed PRF matrix, and
nonpressedsolidPRFwerexedin4%formaldehydefor24h
forhistologicalanalysis.
Liquid platelet‑rich fibrin and exudate processing
The7thtube for eachdonorwasusedfor the preparation of
liquidPRF.Aftercentrifugation,thetubecontainedtwophases:
a yellow‑orange upper phase (liquid‑PRF) and a lower red
phase(redbloodfraction)[Figure2].Understerileconditions,
theliquid‑PRFtubewascarefullyopened,andtheupperphase
wascollectedusingapipette.Thisphasewastransferredtoa
6‑wellplateandincubatedat37°for30min[Figure1].
Growth factor release
ForGFreleasedetermination,thesolidnonpressedPRFmatrix,
plug, and pressed PRF matrix were immediately covered
by 5 ml of cell culture media (RPMI) supplemented with
streptomycin/ampicillin antibiotics and incubated in a cell
cultureincubatorat37°CwithCO2.Thereafter,supernatants
werecollectedafterdenedtimepointsasdescribedbelow.
Liquid‑PRF and the exudates of the pressed PRF‑matrix
andplugswere rst incubated for 30 minat37°Ctoverify
theirabilityofclotformation.Fivemillilitersofcellculture
media (RPMI) supplemented with streptomycin/ampicillin
antibioticswasaddedtotheexudates.Thesupernatantswere
collectedafter 1 h,7h,and 1, 2,7,and10 days andstored
at−80°CforGFquantication.
Enzyme‑linked immunosorbent assay
GF concentrations of vascular epithelial GF (VEGF),
transformingGF‑beta1 (TGF‑β1),andepidermalGF (EGF)
werequantiedusingELISAkits(Douset®ELISA,RandD
Systems,Minneapolis,USA)accordingtothemanufacturer’s
instructions and as previously described.[12] The assay was
performedintriplicateforeachdonorandevaluationgroup.
Thesupernatantswere diluted 1:10 prior to performingthe
measurementforTGF‑ß1andEGF.Theopticaldensitywas
measuredusing amicroplatereader (Innite®M200,Tecan,
Grödig,Austria) set at 450 nm and corrected at 570 nm.
GraphPadPrism7(GraphPadSoftware,Inc.,LaJolla,USA)
wasusedtocalculatethenalGFconcentration.
Statistical analysis
Theresultsareexpressedasthemeansandstandarddeviation.
Figure 2: Macroscopic picture of the platelet-rich fibrin preparation: (a) nonpressed platelet-rich fibrin matrix, (b) pressed platelet-rich fibrin matrix,
(c) platelet-rich fibrin plug, (d) plug exudate, (e) liquid platelet-rich fibrin immediately after centrifugation
d
c
b
a
e
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018 11
GraphPadPrism7(GraphPadSoftware,Inc.,LaJolla,USA)
wasused togeneratechartsand performstatisticalanalyses
using two‑way analysis of variance with Tukey’smultiple
comparisons test (α =0.05). Values were considered as
signicantif P <0.05(*)andhighlysignicantat P <0.01(**),
P <0.001(***),and P <0.0001(****).
Histological preparation
Histological preparation was performed as previously
described.[8]Briey,thesampleswerecutin transversaland
sagittaldirectionsanddehydratedinaseriesofalcoholwith
increasing concentration, treated with xylol, and rinsed in
parafnusinganautomatedprocessor.Thereafter,thesamples
were embedded in paraffin, and four sections (3–4 µm)
of each sample were cut using a rotator microtome.After
deparafnization and rehydration in xylol and alcohol, the
sampleswere stainedwithhematoxylin andeosinandAzan
accordingtoestablishedprotocolsaspreviouslydescribed.[16]
Immunohistochemical staining for CD61, as a marker for
platelets,andCD45,asamarkerforleukocytes,wasperformed
as previously described.[12] The deparafnized slides were
rinsedinacitrate buffer(pH =6)at96°Cfor 20min.After
washingandsubsequentcooling,theslideswereplacedinan
autostainer(LabVision™Autostainer360,ThermoScientic)
loadedwiththeCD61antibody(Dako)ataconcentrationof
1:50or CD45 antibody (Dako) at a concentration of 1:100
and UltraVision™Quanto Detections System horseradish
AEC (peroxidase 3‑amino‑9‑ethylcarbazole) for CD61 and
DAB (3,3’‑ diaminobenzidine techtrachloride) for CD 45.
CounterstainingwasperformedusingHemalum.
Forhistologicalevaluation,alightmicroscope(NikonEclipse
80i,Tokyo,Japan)equippedwithascanningtableconnected
toa PCwithNIS‑Elementssoftware(Nikon,Tokyo,Japan)
was used, and a camera was used to obtain micrographs
and total scans. Total scanning is a method to digitalize
histological slides.A total scan consists of 50–100 single
imagesautomaticallycapturedandmergedtoobtainacomplete
digitalizationoftheregionofinterest.
results
Macroscopic observation
AfterincubationoftheliquidPRFandexudatesfor30min,
structuralchangeswereobserved.LiquidPRFformedaclot.
Incontrast,theexudates ofpressedPRFmatricesand plugs
didnotformanyclotsandmaintainedtheliquidcondition.
Histological analysis
Fibrin structure
All evaluations were performed using longitudinal and
transversalslices.
The structure of the nonpressed solid PRF matrix showed
homogeneousporousstructurewithalargerinterbrousspace
comparedtotheothertwosolidPRFgroups,i.e.,plugand
pressedPRFmatrices[Figure3aanda1].
The pressed PRF matrix showed homogeneous porosity
throughoutthe matrix body.Thebrin structure was dense
andincludedsmallerinterbrousspaceswhencomparedto
thenonpressedPRFmatrix[Figure3bandb1].
Qualitativeevaluationinlongitudinalandtransversalsections
throughoutthe plugshowedaspecic porositypattern.The
centralregionexhibited a rather dense brin structurewith
small interbrous spaces. The peripheral regions showed a
rather porous structure compared to the dense plug body.
However, in general, theinterbrous space in the plug is
smaller than that of the nonpressed PRF matrix [Figure 3c
andc1].
Cellular distribution
The nonpressed PRF matrix showed an even distribution
of platelets (CD61‑positive cells) throughout the
matrix [Figure4a1‑a3]. The dense brin structure in the
pressedPRFmatrixincludedaccumulatedplateletclustersthat
weredistributedthroughoutthematrix[Figure4b1‑b3].The
plugshowedanetofaccumulatedplateletsevenlydistributed
throughouttheplug body over the dense and ratherporous
fibrin structure [Figure 4c1‑c3]. No differences between
theupper,middle,orlower partsofthesolid matriceswere
observed.
Thehomogeneousdistributionofleukocytes(CD45‑positive
cells)was detected in allevaluated groups.Ahigh number
ofthesecellswereobservedthroughoutthenonpressedPRF
Figure 3: Histological micrographs: (a) A total scan of a nonpressed
platelet-rich fibrin (a1) The porous structure of nonpressed platelet-rich
fibrin matrix in Azan; ×100 magnification; scale bar = 100 µm. (b)
A total scan of a pressed platelet-rich fibrin matrix (b1) The dense
homogeneous structure of the pressed platelet-rich fibrin matrix in
Azan; ×100 magnification; scale bar = 100 µm. (c) A total scan of
a platelet-rich fibrin plug (c1) The dense structure (**) and peripheral
porous structure (*) of the platelet-rich fibrin plug in Azan; ×100
magnification; scale bar = 100 µm. (a-c) Are stained using H and E,
×40 magnification: scale bar = 1 mm
c
b
a
c1
b1
a1
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018
12
matrices.ThroughoutthepressedPRFmatrix,leukocyteswere
entrappedwithinthedensebrinstructure.Theplugincluded
evenlydistributedleukocytesthroughoutthedenseandporous
brinstructure[Figure4].
Growth factor release
The quantified GF release was analyzed in the solid
groups(solid nonpressed PRF,PRF plug,and pressed PRF
matrices) and the liquid groups (liquid PRF, plug exudate,
andpressedmatrixexudate)foreachtimepointaswellasthe
accumulatedreleaseover10days.
Vascular endothelial growth factor
TheinitialVEGFreleaseafter1and7hwassimilarinallthe
evaluatedsolidgroups;nostatisticallysignicantdifferences
wereobservedatthesetimepoints.After1and2days,the
PRFplugreleasedahigherconcentrationofVEGFcompared
tothenonpressed PRF‑matrix and the pressed PRF‑matrix.
However, this differencewas not statistically signicant.
Compared to the pressed PRF matrix, the VEGF release
after7 dayswassignicantly higherinthenonpressedPRF
matrix(P<0.01)andthePRFplug(P<0.0001).However,no
statisticallysignicantdifferencewasobservedbetweenthe
nonpressedPRFmatrixandthepressedPRFplug.Onday10,
similarreleasetendenciesasday7wereobserved.However,
nostatisticallysignicantdifferencewasdetectedbetweenthe
evaluatedgroups[Figure5a].
Withintheliquidgroups,noVEGFwasdetectedintheplug
andpressedmatrixexudatesatanytimepoint,whereasliquid
PRF released signicantly higher VEGF concentrations at
thesetimepointscomparedtotheplugexudate(P<0.0001)
andthepressedmatrixexudate(P<0.0001).Startingfromday
1today10,noVEGFwasreleasedfromtheplugandpressed
PRFexudates.Incontrast,liquidPRFreleasedasignicantly
higherVEGFconcentrationondays1,2,7,and10(P<0.0001
foralltimepoints)[Figure5b].
TheinitialGFreleaseoftheliquidPRFafter1 and7 hwas
signicantlyhigherthanthenonpressedPRFmatrix(P<0.01
after 1 h and P < 0.0001 after 7 h), the pressed PRF
matrix(P<0.05after1hand P <0.001after7h),andthePRF
plug(P<0.01after1hand P <0.001after7h).However,with
thetimecourse,theaccumulatedVEGFreleaseover10days
wassignicantly higher inallsolid groups comparedtothe
liquidPRF(nonpressedPRFmatrix, P <0.0001;pressedPRF
matrix, P <0.001;andPRFplug, P <0.0001)[Figure5c].
The accumulated VEGF release for the solid PRF groups
showedthatafter10days,thePRFplugreleasedsignicantly
higherVEGFconcentrationcomparedtothenonpressedRPF
matrix(P<0.0001)andpressedPRF matrix (P < 0.0001).
Nostatisticallysignicant difference was detected between
the nonpressed PRF matrix and the pressed PRF matrix
[Figure5candTable1].
Transforming growth factor
WithinthesolidPRFgroups,theTGF‑β1releasewassimilarin
allgroupsafter1h.Nostatisticallysignicantdifferencewas
observedatthistimepoint.From7hto2days,theTGF‑β1
release in the pressed PRF matrix was frequently higher
than that in the nonpressed PRF matrix and the PRF plug.
However,thisdifferencewasnotstatisticallysignicant.On
day7,thenonpressedPRFmatrixandthePRFplugreleased
signicantlyhigherTGF‑β1releasecomparedtothepressed
PRFmatrix(P<0.05forboth).Asimilarreleasepatternwas
observedonday10;thus,thenonpressedRPFmatrixandthe
PRFplugreleasedsignicantlyhigherTGF‑β1comparedto
thepressedPRFmatrix(P<0.001forboth)[Figure6a].
The matrix and plug exudates released low TGF‑β1
concentrationsonlyafter1and7h.Theplugexudatereleased
signicantly higher TGF‑β1 than the matrix exudate after
1 h (P < 0.05). Liquid PRF released signicantly higher
TGF‑β1than the matrixexudateafter 1 h(P<0.0001) and
7 h (P < 0.0001). Similarly,the TGF‑β1 was signicantly
Figure 4: (a and a1) CD61-marked platelets in the solid nonpressed platelet-rich fibrin matrix. (a2) leukocyte distribution in nonpressed platelet-rich
fibrin matrix marked by CD45 (a3) Platelets in x400 magnification (a4) leukocytes in x400 magnification. (b and b1) CD61-marked platelets in the
solid-pressed platelet-rich fibrin matrix (b) Transversal slices of the pressed platelet-rich fibrin matrix oriented from the upper to the lower region. (b2)
leukocyte distribution in pressed platelet-rich fibrin matrix marked by CD45 (b3) Platelets in x400 magnification (b4) leukocytes in x400 magnification. (c
and c1) CD61-marked platelets in the platelet-rich fibrin plug. (c2) leukocyte distribution in nonpressed platelet-rich fibrin matrix marked by CD45 (c3)
Platelets in x400 magnification (c4) leukocytes in x400 magnification. (a-c) total scan in ×40 magnification; scale bar = 1 mm (a1 and a2), (b1 and
b2), and (c1 and c2) ×100 magnification; scale bar = 100 µm
c
b
ac1
c2
b1
b2
a1
a2
a3
a4
b3
b4
c3
c4
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018 13
higher than the plug exudate after 1 h (P < 0.0001) and
7h (P<0.0001).From1to10days,noTGF‑β1wasdetectedin
bothexudates.However,theliquidPRFreleasedsignicantly
higherTGF‑β1thanbothexudates(P<0.001fordays1and
10; P <0.0001fordays2and7)[Figure6b].
TheinitialTGF‑β1releaseoftheliquidPRFwassimilarto
thatofallsolidgroups.Nostatisticallysignicantdifferences
werefoundafter1and7h.
TheTGF‑β1accumulatedover10daysshowednostatistically
signicant difference between the evaluated solid groups.
These results were, however, all significantly higher
than the accumulated TGF‑β1 release of liquid PRF after
10days(P<0.0001forall)[Figure6c].
Epidermal growth factor
The EGF release in the solid PRF matrices was higher in
the pressed PRF‑matrix and plug compared to that in the
nonpressedPRF‑matrixafter1h.However,this difference
was not statistically significant. After 7 h, the pressed
PRF matrix released significantly higher EGF than the
nonpressed PRF‑matrix (P < 0.0001) and the pressed
PRF plug (P < 0.01).After 1 and 2 days, no statistically
signicantdifferencewasdetectedbetween thegroups.On
day 7, the nonpressed PRF‑matrix released signicantly
higher EGF than the pressed PRF‑matrix (0.05).At this
timepoint,no statistically signicantdifferencewasfound
betweenthenonpressedPRF‑matrixandthePRFplug.After
10 days, a similar release pattern to day 7 was observed.
Table 1: The accumulated growth factor release over 10
days
Sample VEGF (pg/ml) TGF‑β1 (pg/ml) EGF (pg/ml)
Nonpressed
matrix
585.08±64.61 51,184.57±5620.13 2013.75±261.76
Pressed
matrix
529.02±31.57 51,642.46±6522.62 2086.58±346.37
Plug 848.188±132.36**** 54,069.29±7860.99 2025.84±75.08
LiquidPRF 403.82±48.13 26,128.60±9121.23 1375.17±510.07
Matrix
exudate
01351.47±367.16 13.35±6.67
Plugexudate 03617.06±875.17 12.05±5.07
Statisticalanalysisoftheaccumulatedgrowthfactorreleaseafter10days
inallevaluatedgroups.Onlythegroupthatissignicantlyhigherthan
allothergroupsisindicated(****P<0.0001).PRF:Platelet‑richbrin,
VEGF:Vascularendothelialgrowthfactor,TGF‑β1:Transforminggrowth
factor‑β1,EGF:Epidermalgrowthfactor
Figure 5: (a) the growth factor release per time point in the solid PRF
matrices (b) the growth factor release per time point in the liquid PRF
matrices and the exudates (c) the accumulated growth factor release
over 10 days
b
c
a
Figure 6: (a) the growth factor release per time point in the solid PRF
matrices (b) the growth factor release per time point in the liquid PRF
matrices and the exudates (c) the accumulated growth factor release
over 10 days
b
c
a
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018
14
The nonpressed PRF‑matrix released signicantly higher
EGFthanthepressedPRF‑matrix(P<0.01).Similarly,the
PRFplugreleasedsignicantlyhigherEGFthanthepressed
PRF‑matrix(P<0.05).Nostatisticallysignicantdifference
was found between the PRF plug and the nonpressed
PRF‑matrix[Figure7a].
TheEGFreleaseintheplugandpressedPRF‑matrixexudates
wasmarkedlylow,butdetectableafter1and7h.Atthesetime
points,liquidPRFreleasedsignicantlyhigherEGFthanthe
matrixandtheplugexudate(P<0.0001forbothgroupsand
timepoints).Fromdays1to10,noEGFwasdetectedinthe
matrix and plug exudates. The release of EGF from liquid
PRFdecreasedwiththetimecourse,andthisdifferencewas
statistically signicantly higher than the matrix and plug
exudatesonday1 (P < 0.0001), day 2 (P < 0.01), and day
7(P<0.01).Nostatisticallysignicantdifferencewasfound
onday10[Figure7b].
The initial EGF release after 1 h was signicantly higher
in the liquid PRF compared to that in the nonpressed
PRF‑matrix(P<0.05), pressedPRF‑matrix(P<0.05),and
PRFplug(P<0.05).TheaccumulatedEGFreleaseafter7h
and1 and2days wassignicantlyhigher intheliquid PRF
comparedtothatinthe nonpressed PRF‑matrix (P < 0.001
for all) and the PRF plug (P < 0.01 for all), whereas no
statisticallysignicantdifferencewasfoundbetweenpressed
PRF‑matrixandtheliquidPRF.After7days,nostatistically
significant difference was found between the evaluated
groups.After 10 days, the accumulated EGF release was
signicantly higher in all solid PRF groups compared to
thatintheliquidPRF(P<0.01fornonpressedPRF‑matrix,
P <0.001forpressedPRFmatrix,and P <0.01forthePRF
plug).Nostatisticallysignicantdifferencewasfoundforthe
accumulated EGF release between the solid groups on day
10[Figure7c].
dIscussIon
The clinical requirements for a bioactive scaffold include
a stable structure to maintain space and enable cellular
migration of the host tissue, a reservoir of GFs to trigger
the vascularization and regenerative cells, as well as the
degradationofthescaffoldinconcertwiththeregeneration
process.[17] PRF, as a bioactive blood concentrate, is used
in main indication fields in terms of tissue engineering
andregenerativeoralsurgery.[18]Intheeldofperiodontal
regeneration,pressedPRF‑matricesare widelyusedforthe
regeneration of soft‑tissue and recession coverage either
alone or in combination with biomaterials.[19] In addition,
PRFplugspreparedbypressingthePRF‑matrixarewidely
appliedfor socket preservation after tooth extraction.[13] In
thiscontext,thereweresomeconcernsofwhetherprocessing
PRF by pressure may damage the brin structure, destroy
theincluded cells,orwhethertheforcedeliminationof the
matrixexudatewouldlead toalossofthe GFs.Aprevious
studydemonstratedthatthebrinarchitecturehas a major
impact on the released GFs in different blood concentrate
systems.[20]Thereby,differenttechniques were proposedto
standardizethepreparationofPRFandobtainapressedPRF
matrixshowingthatthepreparationmethodsofthePRF‑based
matricessignicantlyinuencethestructureandbioactivity
oftheobtainedpressedPRF‑matrixintermsofGFrelease.[21]
Theultimateaimofthesedevices was to obtain processed
PRFmatricesthataresuitableforclinicalapplicationusing
astandardizedmethod,enablingthepreservationoftheGFs
within the pressed PRF‑matrix or PRF plug, the retention
oftheplateletsandleukocytesinthebrinscaffold,andthe
maintenance of the three‑dimensional brin structure.[15,21]
Toourknowledge,nostudieshaveevaluatedtheeffectofa
standardizedpreparationtechniqueonthepressedPRF‑matrix
andplug compared tononpressedPRF‑matrix.Inaddition,
thereis no informationabouttheGF releaseofliquidPRF
comparedto the PRFmatrixandplug exudates.Therefore,
thepresentstudyfocusedontheanalysisofkeyGFs(VEGF,
EGF, and TGF‑ß1) in solid PRF‑matrices processed by
homogeneouspressure,theirexudates,andliquidPRF.
Thehistologicalanalysisrevealedthatthebrinstructureinthe
pressedPRFmatrixwasdenserthanthatofthenonpressedPRF
matrix.However,thestructurewashomogeneousthroughoutthe
matrixandincludedevenlydistributedleukocytesandplatelets,
Figure 7: (a) the growth factor release per time point in the solid PRF
matrices (b) the growth factor release per time point in the liquid PRF
matrices and the exudates (c) the accumulated growth factor release
over 10 days
c
b
a
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018 15
asdemonstratedbythehistologicalanalysisoflongitudinaland
transversalslices.Thishomogeneousstructureisimportantfor
theclinicalapplicationofthepressedPRFmatrixwhenusedin
periodontaltreatmentorasawounddressing.Thebrinstructure
in the plug showed different porosity areas. The peripheral
part was similar to the nonpressed PRF matrix, whereas the
central part was denser.However, the distribution pattern of
leukocytes and platelets was homogeneous throughout the
plug.Inarecent in vivo study,wedemonstratedthataporous
brinstructure inPRFenables rapidvascularizationandhost
cellpenetration,whileadensebrinstructureisratherresistant
to host cell penetration and vascularization.[16]Translating
theseobservationstotheboneregenerationintermsofsocket
preservation,theperipheralporouspartmaytriggerosteoblasts
andvesselstowardthecentralregion,whilethecentraldense
structuremayserveasaplaceholder.
TheGFconcentrationsoftheinvestigatedsolidPRFmatrices
showed that processing PRF matrices using homogeneous
pressure does not negatively affect the release ofVEGF,
TGF‑ß1,and EGF over 10days.Thereby,the accumulated
GF release showed no statistically significant difference
between the pressed and nonpressed matrices in EGF and
TGF‑ß1.Interestingly,theaccumulatedreleaseofVEGFwas
signicantlyhigherinthepressedPRFplugcomparedtothe
pressedPRFandnonpressedPRFmatrices.These different
releaseprolesoftheinvestigatedGFsarenoteworthy.Fibrin
as the main scaffold of PRF matrices possesses different
binding afnities to GFs.[22] Particularly,VEGF binds to
brinandenablesslowandsustainedreleasepattern.[23]The
compromisedanddensestructureofthePRFplugmayhave
entrappedVEGFwithinits central region and resulted ina
higherandsustained release.Anotherinterestingndingof
thepresentstudyisthatonday7,allevaluatedGFsshowed
signicantlyhigherreleasefromthenonpressedPRFmatrix
andthePRFplugcomparedwiththat fromthepressedPRF
matrix. These observations are likely caused by the matrix
volumeandsurfaceexposure,asthenonpressedPRFmatrix
andtheplughaveahighervolumeandalessexposedsurface
thanthe pressed PRFmatrix.Thisnding is alsosupported
by the fact that the pressed PRF‑matrix released higher
EGF concentration than the other solid groups at the early
timepoints,i.e.,after 1hor1day,butasignicantlylower
concentrationonday7.However,therewerenostatistically
signicantdifferencesintheaccumulatedEGFconcentrations
after10days.Thisndingshowsthatitispossibletoinuence
the release profile of EGF by changing the volume and
structureofPRFbyhomogeneouspressure.EGFisinvolved
in the process of neoangiogenesis.[24] Enhancing the EGF
concentration on early time points may accelerate wound
healingbytriggeringcellsthatareessentialforangiogenesis
andshorteningthetimeperiodofhypoxiaaftertheintended
bloodvesselinterruptionbysurgicalintervention.
In addition, the present study demonstrated that the GF
concentrations of the evaluated solid PRF matrices were
actively released, even after processing by homogeneous
pressure. In contrast, the plug and pressed matrix exudates
showedapassiveGFrelease,whichwasonlydetectedafter
1 and 7 h for TGF‑ß1 and EGF, whereas VEGF was not
detectedwithin the exudates. This effectis in concert with
theobservationthattheexudatesdidnotclotafterincubation
for30min.Thesendingssuggestthattheexudatesofsolid
PRF obtained by its pressure do not include brinogen or
platelets.Similarobservationswerefoundinan in vitro study
that demonstrated no clot formation of the PRF exudate,
evenafterthe addition of bovinethrombin,whichindicates
alackofplateletsandbrinogenswithinthePRFexudate.[25]
Thereby,theevaluationoftheexudatesinthisstudyrepresents
an indicator of the loss of GF during processing the solid
PRFmatricesusing homogenous pressure, whichisashere
demonstratedpracticallyverylow.Inaddition,theuseofthe
exudatesinthisstudyallowedacomparativeillustrationofthe
activeand passiveGFrelease andtherelevanceof platelets
andleukocytesfortheactiveGFrelease.
However, the investigated liquid PRF showed signicantly
higherGF release comparedtothatof theplugandpressed
matrixexudates.Thereleaseprole ofVEGF,TGF‑ß1, and
EGFshowed an active release over 10days, indicating the
viabilityoftheincludedplateletsandleukocytes.Furthermore,
liquidPRFformedaclotafterincubationfor30min.Therefore,
the present observation illustrates that liquid PRF releases
differentGFsover10days.Thisndingiscomplementaryto
theresultsofaprevious in vitro studyshowinghighplatelet
andleukocytenumbersinliquidPRFanditsabilitytorelease
differentGFsafter1h.[12]Thereby,thecombinationofliquid
PRFwithbiomaterialstoenhancetheirbioactivityisclinically
more relevant than the exudate released from solid PRF
matrices.However,controlledclinical studiesareneeded to
furtherevaluatetheclinicalbenetofthissystem.
When comparing liquid PRF with the solid PRF matrices,
the present results suggest that liquid PRF releases high
concentrationsofGFsintheearlytimepoints,whereassolid
PRF matrices release significantly higher concentrations
of GFs after 10 days. This effectmight be related to the
quality and properties of the formed fibrin clot and its
afnitytotheincludedGFs.Inaddition,thevolumeofliquid
PRF(approximately2ml)islessthanthevolumeofthesolid
PRF matrix.Another factor is the coagulation cascade; in
solidPRF,thecoagulationcascadestartsduringcentrifugation,
whilethe coagulation of liquid PRFstarts minutes after its
centrifugation,thustheboostofreleaseismeasureddirectly
afterorduringcoagulationintheearlytimepointsafterplatelet
activation. Platelets activation enhances their capacity to
releaseGFs.[26]Inthiscontext,thecombinationofliquidPRF
andsolidPRFmayprovideafurtherclinicalbenet.However,
controlledclinicalstudiesareneededtofurtherinvestigatethis
application,asitisstillundeterminedwhatconcentrationofa
GFisneededtoregenerateaspecicdefectsize.
Altogether,theresultspresentedinthepresentstudyprovide
afurtherunderstandingoftheclinicallyusedsolidandliquid
PRFmatrices.Thesendingscanhelpclinicianstoconsciously
Al-Maawi, et al.: Homogeneous pressure signicantly enhances VEGF release in the solid PRF plug
International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018
16
apply PRF matrices as a drug delivery system to support
regenerationindifferentindications.
conclusIon
Thepresentstudydemonstratedthelikelihoodofinuencing
the GF release profile in solid PRF matrices by using
homogeneouspressure.The accumulated TGF‑ß1 and EGF
release after 10 days showed active and sustained proles
in all examined solid PRF matrices without statistically
signicantdifferences.ThePRF plug released signicantly
higherVEGF over 10dayscomparedtothatof the pressed
PRFandnonpressedPRFmatrices.IncontrasttotheactiveGF
releaseinliquidPRF,thesolidPRFexudatesshowedapassive
GFrelease that was only detected in the early time points.
Interestingly,liquid PRF released signicantly higher GF
concentrationsintheearlytimepoints,i.e.,1h–1day,whereas
theaccumulated GF release over10 days was signicantly
higherinthesolidPRFmatricescomparedtothatinliquidPRF.
Theseresultssuggestthatthecombinationofsolidandliquid
PRFmayprovidesignicantbenetforclinicalapplicationto
obtainsustainedbioactivity.Thesendingsareofgreatinterest
forscientistsandcliniciansandprovidefurtherunderstanding
ofPRFmatricesasadrugdeliverysystem.
Acknowledgment
TheauthorswouldliketothankMrs.VerenaHoffmanforher
excellenttechnicalassistance.
Financial support and sponsorship
Nil.
Conflicts of interest
Therearenoconictsofinterest.
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