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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

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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
Inthelastdecades,differenttechniqueshavebeendeveloped
to promote bone and soft‑tissue regeneration.[1] Natural
biomaterials,suchasbovine‑derivedxenogeneicbiomaterials
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 purication
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‑richbrin(PRF)existsinbothsolidanduidforms.Thepresentstudywasthersttoevaluatetheinuenceofhomogeneous
pressureonthegrowthfactor(GF)releaseinpressedPRF‑matricesandplugs.Methods and Material:AsolidPRF‑matrix(208g;8min)
waspressedtoobtainaplug,andapressedPRF‑matrixthatareusedinclinicalapplication.Thereleasedexudateswereevaluatedcomparedto
liquidPRF(60gand3min).TheVEGF,TGF‑ß1andEGFreleasewasquantiedusingELISA.Thebrinstructureandcellularcomponentsin
solidPRFgroupswereevaluatedhistologically.Results:ThepressedPRF‑matrixandPRF‑plugexhibiteddenserbrinstructurecomparedto
thenon‑pressedPRF‑matrix.Onday7,thePRF‑plugandnon‑pressedPRF‑matrixshowedsignicantlyhigherreleaseofVEGF,TGF‑ß1and
EGFcomparedtothatofthepressedPRF‑matrix.TheaccumulatedVEGFconcentrationwassignicantlyhigherinthePRF‑plugcompared
tothatinthePRF‑matrixandnon‑pressedPRF‑matrix.TheaccumulatedEGFandTGF‑ß1concentrationsover10daysshowednostatistically
signicantdifferencesbetweentheevaluatedsolidPRFgroups.TheexudatesreleasedTGF‑ß1andEGFpassively,thatwasonlydetectable
inafter1and7hours.LiquidPRFreleased signicantlyhigherGFsthantheexudatesatallinvestigatedtimepoints.TheearlyVEGFand
EGFrelease inliquidPRF (1 hourto1 day)wassignicantly higherthanthat in thesolidPRF‑matrices. Onday10, signicantlyhigher
accumulatedGFsweredetectedinthesolidPRFgroupscomparedtothoseintheliquidPRF.Thus,thecombinationofbothsolidandliquid
PRFisapotentialtooltogenerateaclinicallyrelevantsystemwithsustainedbioactivity.Conclusions:Theseresultshighlightthepotential
toinuencetheGFsreleaseproleofsolidPRFmatricesbypressureandobtainaclinicallyapplicableplugwithsignicantlyhigherVEGF
release,providingfurtherunderstandingofthereleaseproleofPRFmatricesasadrugdeliverysystem.
Keywords:Growthfactors,liquidplatelet‑richbrin,low‑speedcentrifugationconcept,regeneration,solidplatelet‑richbrin
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
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DOI:
10.4103/GFSC.GFSC_9_18
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How to cite this article: Al‑Maawi S, Herrera‑VizcainoC, Dohle E,
ZrncTA,ParviniP,Schwarz F,et al.Homogeneous pressure inuences
thegrowthfactorreleaseprolesinsolid platelet‑richbrinmatricesand
enhancesvascularendothelialgrowthfactorreleaseinthesolidplatelet‑rich
brinplugs.IntJGrowthFactorsStemCellsDent2018;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 signicantly 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
aminimally invasiveautologousregenerationsource, blood
concentrateshave beenintroducedasa promisingclinically
relevantmethod.Bloodconcentrates aregeneratedfromthe
patients’ownperipheralblood,concentratedbycentrifugation,
andcanreleasedifferentgrowthfactors(GFs).[4]
Therst‑generationbloodconcentrates, termedplatelet‑rich
plasma, require multiple centrifugations and the addition
of anticoagulants and external chemical activation
substrates.[5]However,thiselaboratepreparationmayprovide
majordrawbacksforclinicalroutine.Thus,theintroductionof
platelet‑richbrin(PRF)facilitatestheclinicalapplicationof
bloodconcentratesystems.PRFisobtainedfromthepatients’
venous blood by single centrifugation without additional
anticoagulants,makingita100%autologousbloodconcentrate
system.[6]Thefast and easy preparationof PRF makes this
materialclinicallyapplicableandfavorableforclinicians.[7]
During centrifugation, a solid‑brin matrix is generated by
theactivationoftheplateletsthrough their interaction with
the tube surface.[8] The activation of platelets promotes the
release of different GFs.[9] The resultingsolid PRF matrix
consistsofabrinscaffoldthatincludesplatelets,leukocytes,
and plasma proteins. The preparation protocol of the rst
describedsolidPRFrequirestheapplicationofahighrelative
centrifugationforce(RCF).[8]TheroleoftheappliedRCFand
centrifugationtimeintheformation,structure,andbioactivity
ofPRFmatriceshas beenextensivelystudiedbyour group.
Arecent exvivo study reported a protocol modication by
reducingtheappliedRCFfollowingtheso‑calledlow‑speed
centrifugation concept (LSCC), resulting in a more porous
brinmatrixthatcontainedahighernumberofplateletsand
leukocytescomparedtosolidPRFprepared byusingahigh
RCF.[10] Further protocol adjustment to maintain the RCF
andpropose a reduction ofthe centrifugation time ledto a
signicantincreaseinthereleaseofGFs,particularlyvascular
endothelial GF (VEGF) measured in vitro.[11]Moreover,
reducingtheRCFinuencedtheplateletdistributionpattern
throughoutthematrix.ThesolidPRFmatrixpreparedusing
a reduced RCF showed more evenly distributed platelets
comparedtotheaccumulatedplateletsatthebottomofsolid
PRFmatricespreparedusingahighRCF.[11]
InadditiontothesolidPRF,theclinicalneedsforaliquidPRF
requiredthedevelopmentofaliquidPRFwithoutadditional
anticoagulants. Following the LSCC, a liquid PRF rich in
platelets,leukocytes,andGFswasachievedbyasystematical
reductionoftheappliedRCFandcentrifugationtime.[12]The
introductionoftheliquidPRFwidenedtherangeofthePRF
applicationinsurgicalperiodontologicaltherapyandfacilitated
itscombinationwithbiomaterials.
Intheclinicalapplication,solidPRFisprocessedbypressure
to obtain tailored PRF forms that are suitable for specic
indications. For instance, solid PRF matrices are pressed
togenerateplugs that areusedforsocketpreservation after
teethareextracted.[13]Solid‑pressedPRFmatricesobtainedby
pressingthesolidPRFmatrixareappliedinperiodontology
forrootcoverage,incombinationwithbiomaterialsandasa
wounddressing.[14]Duringthepressureprocess,liquidexudate
isreleasedfromthesolidPRFmatrix.Todate,littleisknown
abouttheinuenceofpressureontheGFrelease ofpressed
PRFmatrixandplugandtheirexudates.Toourknowledge,the
presentstudyisthersttoevaluatetheeffectofastandardized
preparationtechniquetogainpressedPRF matrix and plug
comparedtononpressedPRFmatrix.Theaimofthepresent
studywastoanalyzetheeffectofsolidPRFmatrixprocessing
by pressure on the bioactivity, regenerative capacity, and
structureoftheclinicallyusedPRF plugs and pressed PRF
matricescomparedtothenonpressedsolidPRFmatrices.In
addition,thebioactivityofthereleasedexudateswasevaluated
comparedtothatoftheliquidPRF.
MaterIals and Methods
TheapplicationofPRFinthisstudywasinaccordancewiththe
principleofinformedconsentandapprovedbytheresponsible
EthicsCommissionofthestateofHessen,Germany(265/17).
Platelet‑rich fibrin preparation
ForPRFpreparation,sixdonors(threemalesandthreefemales)
were included in the present study.The preparation was
performedaspreviouslydescribed[Figure1].[8]Briey,after
obtaining informed consent from each of the participating
donors,venousbloodwascollectedfromtheperipheralmedian
cubitalvein.SixtubeswereusedtogeneratesolidPRF(Red
tubes [10 ml], Process for PRF™, Nice, France), and one
tube(Orangetube[10ml],ProcessforPRF™,Nice,France)
wasusedtoobtainliquidPRF.Afterbloodcollection,thetubes
wereimmediatelyplacedinpreprogrammedcentrifuges(Duo
centrifuge,ProcessforPRF™,Nice,France)andcentrifuged
accordingtoestablishedprotocols:
• SolidPRFmatrix: 208g 8min
• LiquidPRFmatrix: 60g 3min.
Solid platelet‑rich fibrin processing
Understerileconditions, the tubesweregentlyopened, and
thePRFmatricesweresubsequentlycarefullyretrievedfrom
the tube and isolated from the red blood fraction without
damagingtheinterfaceofthematrix.APRFboxthatfacilitates
homogeneous pressure application was used to maintain
standardizedconditionsduringprocessing thePRFmatrices
toplugsandpressedPRFmatrices.Foreachdonor,onePRF
matrixwastransferredtothePRFbox(PRFbox,Processfor
PRF™, Nice, France), placed into the preformed hole and
carefullycompressedusing the compressing tool according
to the manufacturer’s instructions as reported elsewhere.[15]
Theresultingplugwastransferredtoa6‑wellplate,andthe
releasedexudatewascollectedandtransferredtoa6‑wellplate
usinga pipette. The secondPRF matrix was placed on the
PRFboxgridandcoveredbythepressuretoolandboxcover
withoutactivepressure.After5min,theboxwasuncovered,
andthepressedPRFmatrixwastransferredtoa6‑wellplate.
Theresultingexudatewascollectedandtransferredtoa6‑well
plate.The third PRF matrixwasdirectlyplacedina 6‑well
plateandusedasanonpressedcontrol[Figure2].Thesame
procedurewas repeatedwiththree additionalPRFmatrices,
Figure 1: Flowchart of the study design
Al-Maawi, et al.: Homogeneous pressure signicantly 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
nonpressedsolidPRFwerexedin4%formaldehydefor24h
forhistologicalanalysis.
Liquid platelet‑rich fibrin and exudate processing
The7thtube for eachdonorwasusedfor the preparation of
liquidPRF.Aftercentrifugation,thetubecontainedtwophases:
a yellow‑orange upper phase (liquid‑PRF) and a lower red
phase(redbloodfraction)[Figure2].Understerileconditions,
theliquid‑PRFtubewascarefullyopened,andtheupperphase
wascollectedusingapipette.Thisphasewastransferredtoa
6‑wellplateandincubatedat37°for30min[Figure1].
Growth factor release
ForGFreleasedetermination,thesolidnonpressedPRFmatrix,
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
cultureincubatorat37°CwithCO2.Thereafter,supernatants
werecollectedafterdenedtimepointsasdescribedbelow.
Liquid‑PRF and the exudates of the pressed PRF‑matrix
andplugswere rst incubated for 30 minat37°Ctoverify
theirabilityofclotformation.Fivemillilitersofcellculture
media (RPMI) supplemented with streptomycin/ampicillin
antibioticswasaddedtotheexudates.Thesupernatantswere
collectedafter 1 h,7h,and 1, 2,7,and10 days andstored
at−80°CforGFquantication.
Enzyme‑linked immunosorbent assay
GF concentrations of vascular epithelial GF (VEGF),
transformingGF‑beta1 (TGF‑β1),andepidermalGF (EGF)
werequantiedusingELISAkits(Douset®ELISA,RandD
Systems,Minneapolis,USA)accordingtothemanufacturer’s
instructions and as previously described.[12] The assay was
performedintriplicateforeachdonorandevaluationgroup.
Thesupernatantswere diluted 1:10 prior to performingthe
measurementforTGF‑ß1andEGF.Theopticaldensitywas
measuredusing amicroplatereader (Innite®M200,Tecan,
Grödig,Austria) set at 450 nm and corrected at 570 nm.
GraphPadPrism7(GraphPadSoftware,Inc.,LaJolla,USA)
wasusedtocalculatethenalGFconcentration.
Statistical analysis
Theresultsareexpressedasthemeansandstandarddeviation.
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
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International Journal of Growth Factors and Stem Cells in Dentistry ¦ Volume 1 ¦ Issue 1 ¦ January-April 2018 11
GraphPadPrism7(GraphPadSoftware,Inc.,LaJolla,USA)
wasused togeneratechartsand performstatisticalanalyses
using two‑way analysis of variance with Tukey’smultiple
comparisons test (α =0.05). Values were considered as
signicantif P <0.05(*)andhighlysignicantat P <0.01(**),
P <0.001(***),and P <0.0001(****).
Histological preparation
Histological preparation was performed as previously
described.[8]Briey,thesampleswerecutin transversaland
sagittaldirectionsanddehydratedinaseriesofalcoholwith
increasing concentration, treated with xylol, and rinsed in
parafnusinganautomatedprocessor.Thereafter,thesamples
were embedded in paraffin, and four sections (3–4 µm)
of each sample were cut using a rotator microtome.After
deparafnization and rehydration in xylol and alcohol, the
sampleswere stainedwithhematoxylin andeosinandAzan
accordingtoestablishedprotocolsaspreviouslydescribed.[16]
Immunohistochemical staining for CD61, as a marker for
platelets,andCD45,asamarkerforleukocytes,wasperformed
as previously described.[12] The deparafnized slides were
rinsedinacitrate buffer(pH =6)at96°Cfor 20min.After
washingandsubsequentcooling,theslideswereplacedinan
autostainer(LabVision™Autostainer360,ThermoScientic)
loadedwiththeCD61antibody(Dako)ataconcentrationof
1:50or 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.
CounterstainingwasperformedusingHemalum.
Forhistologicalevaluation,alightmicroscope(NikonEclipse
80i,Tokyo,Japan)equippedwithascanningtableconnected
toa PCwithNIS‑Elementssoftware(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
imagesautomaticallycapturedandmergedtoobtainacomplete
digitalizationoftheregionofinterest.
results
Macroscopic observation
AfterincubationoftheliquidPRFandexudatesfor30min,
structuralchangeswereobserved.LiquidPRFformedaclot.
Incontrast,theexudates ofpressedPRFmatricesand plugs
didnotformanyclotsandmaintainedtheliquidcondition.
Histological analysis
Fibrin structure
All evaluations were performed using longitudinal and
transversalslices.
The structure of the nonpressed solid PRF matrix showed
homogeneousporousstructurewithalargerinterbrousspace
comparedtotheothertwosolidPRFgroups,i.e.,plugand
pressedPRFmatrices[Figure3aanda1].
The pressed PRF matrix showed homogeneous porosity
throughoutthe matrix body.Thebrin structure was dense
andincludedsmallerinterbrousspaceswhencomparedto
thenonpressedPRFmatrix[Figure3bandb1].
Qualitativeevaluationinlongitudinalandtransversalsections
throughoutthe plugshowedaspecic porositypattern.The
centralregionexhibited a rather dense brin structurewith
small interbrous spaces. The peripheral regions showed a
rather porous structure compared to the dense plug body.
However, in general, theinterbrous space in the plug is
smaller than that of the nonpressed PRF matrix [Figure 3c
andc1].
Cellular distribution
The nonpressed PRF matrix showed an even distribution
of platelets (CD61‑positive cells) throughout the
matrix [Figure4a1‑a3]. The dense brin structure in the
pressedPRFmatrixincludedaccumulatedplateletclustersthat
weredistributedthroughoutthematrix[Figure4b1‑b3].The
plugshowedanetofaccumulatedplateletsevenlydistributed
throughouttheplug body over the dense and ratherporous
fibrin structure [Figure 4c1‑c3]. No differences between
theupper,middle,orlower partsofthesolid matriceswere
observed.
Thehomogeneousdistributionofleukocytes(CD45‑positive
cells)was detected in allevaluated groups.Ahigh number
ofthesecellswereobservedthroughoutthenonpressedPRF
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
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Al-Maawi, et al.: Homogeneous pressure signicantly 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.ThroughoutthepressedPRFmatrix,leukocyteswere
entrappedwithinthedensebrinstructure.Theplugincluded
evenlydistributedleukocytesthroughoutthedenseandporous
brinstructure[Figure4].
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,
andpressedmatrixexudate)foreachtimepointaswellasthe
accumulatedreleaseover10days.
Vascular endothelial growth factor
TheinitialVEGFreleaseafter1and7hwassimilarinallthe
evaluatedsolidgroups;nostatisticallysignicantdifferences
wereobservedatthesetimepoints.After1and2days,the
PRFplugreleasedahigherconcentrationofVEGFcompared
tothenonpressed PRF‑matrix and the pressed PRF‑matrix.
However, this differencewas not statistically signicant.
Compared to the pressed PRF matrix, the VEGF release
after7 dayswassignicantly higherinthenonpressedPRF
matrix(P<0.01)andthePRFplug(P<0.0001).However,no
statisticallysignicantdifferencewasobservedbetweenthe
nonpressedPRFmatrixandthepressedPRFplug.Onday10,
similarreleasetendenciesasday7wereobserved.However,
nostatisticallysignicantdifferencewasdetectedbetweenthe
evaluatedgroups[Figure5a].
Withintheliquidgroups,noVEGFwasdetectedintheplug
andpressedmatrixexudatesatanytimepoint,whereasliquid
PRF released signicantly higher VEGF concentrations at
thesetimepointscomparedtotheplugexudate(P<0.0001)
andthepressedmatrixexudate(P<0.0001).Startingfromday
1today10,noVEGFwasreleasedfromtheplugandpressed
PRFexudates.Incontrast,liquidPRFreleasedasignicantly
higherVEGFconcentrationondays1,2,7,and10(P<0.0001
foralltimepoints)[Figure5b].
TheinitialGFreleaseoftheliquidPRFafter1 and7 hwas
signicantlyhigherthanthenonpressedPRFmatrix(P<0.01
after 1 h and P < 0.0001 after 7 h), the pressed PRF
matrix(P<0.05after1hand P <0.001after7h),andthePRF
plug(P<0.01after1hand P <0.001after7h).However,with
thetimecourse,theaccumulatedVEGFreleaseover10days
wassignicantly higher inallsolid groups comparedtothe
liquidPRF(nonpressedPRFmatrix, P <0.0001;pressedPRF
matrix, P <0.001;andPRFplug, P <0.0001)[Figure5c].
The accumulated VEGF release for the solid PRF groups
showedthatafter10days,thePRFplugreleasedsignicantly
higherVEGFconcentrationcomparedtothenonpressedRPF
matrix(P<0.0001)andpressedPRF matrix (P < 0.0001).
Nostatisticallysignicant difference was detected between
the nonpressed PRF matrix and the pressed PRF matrix
[Figure5candTable1].
Transforming growth factor
WithinthesolidPRFgroups,theTGF‑β1releasewassimilarin
allgroupsafter1h.Nostatisticallysignicantdifferencewas
observedatthistimepoint.From7hto2days,theTGF‑β1
release in the pressed PRF matrix was frequently higher
than that in the nonpressed PRF matrix and the PRF plug.
However,thisdifferencewasnotstatisticallysignicant.On
day7,thenonpressedPRFmatrixandthePRFplugreleased
signicantlyhigherTGF‑β1releasecomparedtothepressed
PRFmatrix(P<0.05forboth).Asimilarreleasepatternwas
observedonday10;thus,thenonpressedRPFmatrixandthe
PRFplugreleasedsignicantlyhigherTGF‑β1comparedto
thepressedPRFmatrix(P<0.001forboth)[Figure6a].
The matrix and plug exudates released low TGF‑β1
concentrationsonlyafter1and7h.Theplugexudatereleased
signicantly higher TGF‑β1 than the matrix exudate after
1 h (P < 0.05). Liquid PRF released signicantly higher
TGF‑β1than the matrixexudateafter 1 h(P<0.0001) and
7 h (P < 0.0001). Similarly,the TGF‑β1 was signicantly
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 signicantly 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
7h (P<0.0001).From1to10days,noTGF‑β1wasdetectedin
bothexudates.However,theliquidPRFreleasedsignicantly
higherTGF‑β1thanbothexudates(P<0.001fordays1and
10; P <0.0001fordays2and7)[Figure6b].
TheinitialTGF‑β1releaseoftheliquidPRFwassimilarto
thatofallsolidgroups.Nostatisticallysignicantdifferences
werefoundafter1and7h.
TheTGF‑β1accumulatedover10daysshowednostatistically
signicant difference between the evaluated solid groups.
These results were, however, all significantly higher
than the accumulated TGF‑β1 release of liquid PRF after
10days(P<0.0001forall)[Figure6c].
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
nonpressedPRF‑matrixafter1h.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
signicantdifferencewasdetectedbetween thegroups.On
day 7, the nonpressed PRF‑matrix released signicantly
higher EGF than the pressed PRF‑matrix (0.05).At this
timepoint,no statistically signicantdifferencewasfound
betweenthenonpressedPRF‑matrixandthePRFplug.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
LiquidPRF 403.82±48.13 26,128.60±9121.23 1375.17±510.07
Matrix
exudate
01351.47±367.16 13.35±6.67
Plugexudate 03617.06±875.17 12.05±5.07
Statisticalanalysisoftheaccumulatedgrowthfactorreleaseafter10days
inallevaluatedgroups.Onlythegroupthatissignicantlyhigherthan
allothergroupsisindicated(****P<0.0001).PRF:Platelet‑richbrin,
VEGF:Vascularendothelialgrowthfactor,TGF‑β1:Transforminggrowth
factor‑β1,EGF:Epidermalgrowthfactor
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 signicantly 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 signicantly higher
EGFthanthepressedPRF‑matrix(P<0.01).Similarly,the
PRFplugreleasedsignicantlyhigherEGFthanthepressed
PRF‑matrix(P<0.05).Nostatisticallysignicantdifference
was found between the PRF plug and the nonpressed
PRF‑matrix[Figure7a].
TheEGFreleaseintheplugandpressedPRF‑matrixexudates
wasmarkedlylow,butdetectableafter1and7h.Atthesetime
points,liquidPRFreleasedsignicantlyhigherEGFthanthe
matrixandtheplugexudate(P<0.0001forbothgroupsand
timepoints).Fromdays1to10,noEGFwasdetectedinthe
matrix and plug exudates. The release of EGF from liquid
PRFdecreasedwiththetimecourse,andthisdifferencewas
statistically signicantly higher than the matrix and plug
exudatesonday1 (P < 0.0001), day 2 (P < 0.01), and day
7(P<0.01).Nostatisticallysignicantdifferencewasfound
onday10[Figure7b].
The initial EGF release after 1 h was signicantly higher
in the liquid PRF compared to that in the nonpressed
PRF‑matrix(P<0.05), pressedPRF‑matrix(P<0.05),and
PRFplug(P<0.05).TheaccumulatedEGFreleaseafter7h
and1 and2days wassignicantlyhigher intheliquid PRF
comparedtothatinthe nonpressed PRF‑matrix (P < 0.001
for all) and the PRF plug (P < 0.01 for all), whereas no
statisticallysignicantdifferencewasfoundbetweenpressed
PRF‑matrixandtheliquidPRF.After7days,nostatistically
significant difference was found between the evaluated
groups.After 10 days, the accumulated EGF release was
signicantly higher in all solid PRF groups compared to
thatintheliquidPRF(P<0.01fornonpressedPRF‑matrix,
P <0.001forpressedPRFmatrix,and P <0.01forthePRF
plug).Nostatisticallysignicantdifferencewasfoundforthe
accumulated EGF release between the solid groups on day
10[Figure7c].
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
degradationofthescaffoldinconcertwiththeregeneration
process.[17] PRF, as a bioactive blood concentrate, is used
in main indication fields in terms of tissue engineering
andregenerativeoralsurgery.[18]Intheeldofperiodontal
regeneration,pressedPRF‑matricesare widelyusedforthe
regeneration of soft‑tissue and recession coverage either
alone or in combination with biomaterials.[19] In addition,
PRFplugspreparedbypressingthePRF‑matrixarewidely
appliedfor socket preservation after tooth extraction.[13] In
thiscontext,thereweresomeconcernsofwhetherprocessing
PRF by pressure may damage the brin structure, destroy
theincluded cells,orwhethertheforcedeliminationof the
matrixexudatewouldlead toalossofthe GFs.Aprevious
studydemonstratedthatthebrinarchitecturehas a major
impact on the released GFs in different blood concentrate
systems.[20]Thereby,differenttechniques were proposedto
standardizethepreparationofPRFandobtainapressedPRF
matrixshowingthatthepreparationmethodsofthePRF‑based
matricessignicantlyinuencethestructureandbioactivity
oftheobtainedpressedPRF‑matrixintermsofGFrelease.[21]
Theultimateaimofthesedevices was to obtain processed
PRFmatricesthataresuitableforclinicalapplicationusing
astandardizedmethod,enablingthepreservationoftheGFs
within the pressed PRF‑matrix or PRF plug, the retention
oftheplateletsandleukocytesinthebrinscaffold,andthe
maintenance of the three‑dimensional brin structure.[15,21]
Toourknowledge,nostudieshaveevaluatedtheeffectofa
standardizedpreparationtechniqueonthepressedPRF‑matrix
andplug compared tononpressedPRF‑matrix.Inaddition,
thereis no informationabouttheGF releaseofliquidPRF
comparedto the PRFmatrixandplug exudates.Therefore,
thepresentstudyfocusedontheanalysisofkeyGFs(VEGF,
EGF, and TGF‑ß1) in solid PRF‑matrices processed by
homogeneouspressure,theirexudates,andliquidPRF.
Thehistologicalanalysisrevealedthatthebrinstructureinthe
pressedPRFmatrixwasdenserthanthatofthenonpressedPRF
matrix.However,thestructurewashomogeneousthroughoutthe
matrixandincludedevenlydistributedleukocytesandplatelets,
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 signicantly 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
asdemonstratedbythehistologicalanalysisoflongitudinaland
transversalslices.Thishomogeneousstructureisimportantfor
theclinicalapplicationofthepressedPRFmatrixwhenusedin
periodontaltreatmentorasawounddressing.Thebrinstructure
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.Inarecent in vivo study,wedemonstratedthataporous
brinstructure inPRFenables rapidvascularizationandhost
cellpenetration,whileadensebrinstructureisratherresistant
to host cell penetration and vascularization.[16]Translating
theseobservationstotheboneregenerationintermsofsocket
preservation,theperipheralporouspartmaytriggerosteoblasts
andvesselstowardthecentralregion,whilethecentraldense
structuremayserveasaplaceholder.
TheGFconcentrationsoftheinvestigatedsolidPRFmatrices
showed that processing PRF matrices using homogeneous
pressure does not negatively affect the release ofVEGF,
TGF‑ß1,and EGF over 10days.Thereby,the accumulated
GF release showed no statistically significant difference
between the pressed and nonpressed matrices in EGF and
TGF‑ß1.Interestingly,theaccumulatedreleaseofVEGFwas
signicantlyhigherinthepressedPRFplugcomparedtothe
pressedPRFandnonpressedPRFmatrices.These different
releaseprolesoftheinvestigatedGFsarenoteworthy.Fibrin
as the main scaffold of PRF matrices possesses different
binding afnities to GFs.[22] Particularly,VEGF binds to
brinandenablesslowandsustainedreleasepattern.[23]The
compromisedanddensestructureofthePRFplugmayhave
entrappedVEGFwithinits central region and resulted ina
higherandsustained release.Anotherinterestingndingof
thepresentstudyisthatonday7,allevaluatedGFsshowed
signicantlyhigherreleasefromthenonpressedPRFmatrix
andthePRFplugcomparedwiththat fromthepressedPRF
matrix. These observations are likely caused by the matrix
volumeandsurfaceexposure,asthenonpressedPRFmatrix
andtheplughaveahighervolumeandalessexposedsurface
thanthe pressed PRFmatrix.Thisnding is alsosupported
by the fact that the pressed PRF‑matrix released higher
EGF concentration than the other solid groups at the early
timepoints,i.e.,after 1hor1day,butasignicantlylower
concentrationonday7.However,therewerenostatistically
signicantdifferencesintheaccumulatedEGFconcentrations
after10days.Thisndingshowsthatitispossibletoinuence
the release profile of EGF by changing the volume and
structureofPRFbyhomogeneouspressure.EGFisinvolved
in the process of neoangiogenesis.[24] Enhancing the EGF
concentration on early time points may accelerate wound
healingbytriggeringcellsthatareessentialforangiogenesis
andshorteningthetimeperiodofhypoxiaaftertheintended
bloodvesselinterruptionbysurgicalintervention.
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
showedapassiveGFrelease,whichwasonlydetectedafter
1 and 7 h for TGF‑ß1 and EGF, whereas VEGF was not
detectedwithin the exudates. This effectis in concert with
theobservationthattheexudatesdidnotclotafterincubation
for30min.Thesendingssuggestthattheexudatesofsolid
PRF obtained by its pressure do not include brinogen or
platelets.Similarobservationswerefoundinan in vitro study
that demonstrated no clot formation of the PRF exudate,
evenafterthe addition of bovinethrombin,whichindicates
alackofplateletsandbrinogenswithinthePRFexudate.[25]
Thereby,theevaluationoftheexudatesinthisstudyrepresents
an indicator of the loss of GF during processing the solid
PRFmatricesusing homogenous pressure, whichisashere
demonstratedpracticallyverylow.Inaddition,theuseofthe
exudatesinthisstudyallowedacomparativeillustrationofthe
activeand passiveGFrelease andtherelevanceof platelets
andleukocytesfortheactiveGFrelease.
However, the investigated liquid PRF showed signicantly
higherGF release comparedtothatof theplugandpressed
matrixexudates.Thereleaseprole ofVEGF,TGF‑ß1, and
EGFshowed an active release over 10days, indicating the
viabilityoftheincludedplateletsandleukocytes.Furthermore,
liquidPRFformedaclotafterincubationfor30min.Therefore,
the present observation illustrates that liquid PRF releases
differentGFsover10days.Thisndingiscomplementaryto
theresultsofaprevious in vitro studyshowinghighplatelet
andleukocytenumbersinliquidPRFanditsabilitytorelease
differentGFsafter1h.[12]Thereby,thecombinationofliquid
PRFwithbiomaterialstoenhancetheirbioactivityisclinically
more relevant than the exudate released from solid PRF
matrices.However,controlledclinical studiesareneeded to
furtherevaluatetheclinicalbenetofthissystem.
When comparing liquid PRF with the solid PRF matrices,
the present results suggest that liquid PRF releases high
concentrationsofGFsintheearlytimepoints,whereassolid
PRF matrices release significantly higher concentrations
of GFs after 10 days. This effectmight be related to the
quality and properties of the formed fibrin clot and its
afnitytotheincludedGFs.Inaddition,thevolumeofliquid
PRF(approximately2ml)islessthanthevolumeofthesolid
PRF matrix.Another factor is the coagulation cascade; in
solidPRF,thecoagulationcascadestartsduringcentrifugation,
whilethe coagulation of liquid PRFstarts minutes after its
centrifugation,thustheboostofreleaseismeasureddirectly
afterorduringcoagulationintheearlytimepointsafterplatelet
activation. Platelets activation enhances their capacity to
releaseGFs.[26]Inthiscontext,thecombinationofliquidPRF
andsolidPRFmayprovideafurtherclinicalbenet.However,
controlledclinicalstudiesareneededtofurtherinvestigatethis
application,asitisstillundeterminedwhatconcentrationofa
GFisneededtoregenerateaspecicdefectsize.
Altogether,theresultspresentedinthepresentstudyprovide
afurtherunderstandingoftheclinicallyusedsolidandliquid
PRFmatrices.Thesendingscanhelpclinicianstoconsciously
Al-Maawi, et al.: Homogeneous pressure signicantly 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
regenerationindifferentindications.
conclusIon
Thepresentstudydemonstratedthelikelihoodofinuencing
the GF release profile in solid PRF matrices by using
homogeneouspressure.The accumulated TGF‑ß1 and EGF
release after 10 days showed active and sustained proles
in all examined solid PRF matrices without statistically
signicantdifferences.ThePRF plug released signicantly
higherVEGF over 10dayscomparedtothatof the pressed
PRFandnonpressedPRFmatrices.IncontrasttotheactiveGF
releaseinliquidPRF,thesolidPRFexudatesshowedapassive
GFrelease that was only detected in the early time points.
Interestingly,liquid PRF released signicantly higher GF
concentrationsintheearlytimepoints,i.e.,1h–1day,whereas
theaccumulated GF release over10 days was signicantly
higherinthesolidPRFmatricescomparedtothatinliquidPRF.
Theseresultssuggestthatthecombinationofsolidandliquid
PRFmayprovidesignicantbenetforclinicalapplicationto
obtainsustainedbioactivity.Thesendingsareofgreatinterest
forscientistsandcliniciansandprovidefurtherunderstanding
ofPRFmatricesasadrugdeliverysystem.
Acknowledgment
TheauthorswouldliketothankMrs.VerenaHoffmanforher
excellenttechnicalassistance.
Financial support and sponsorship
Nil.
Conflicts of interest
Therearenoconictsofinterest.
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... Platelet-rich fibrin (PRF) is an autologous source of the platelets and growth factors in the form of the fibrin [38][39][40][41]. Due to the slow polymerization throughout centrifugation and fibrin-created construction, PRF is a better healing biomaterial than platelet-rich plasma (PRP) and other fibrin adhesives [42][43][44]. PRF is a rich source of cytokines and growth factors such as insulin-like growth factor-1 (IGF-1), VEGF, transforming growth factor ß1 (TGF-β1), and PDGF [45][46][47][48][49]. The existence of growth factors and cytokines in the PRF and platelets displays an important role in wound healing [34]. ...
... PRF contains necessary cells (monocytes, lymphocytes, and neutrophil), which are important during the wound healing process [54,115]. Neutrophils and macrophages are one of the first cell types find in the wound area, their role also include to phagocytize debris, microbes, and necrotic tissue, thereby preventing infection [42,49]. Also, PRF as a source of autologous growth factors is a key player in wound healing, tissue regeneration, new blood vessel formation, and prevention of infection [42,49,117]. ...
... Neutrophils and macrophages are one of the first cell types find in the wound area, their role also include to phagocytize debris, microbes, and necrotic tissue, thereby preventing infection [42,49]. Also, PRF as a source of autologous growth factors is a key player in wound healing, tissue regeneration, new blood vessel formation, and prevention of infection [42,49,117]. Additionally, the controlled and sustained release of proteins from the Gel-CH/CH-PRF hydrogel (Fig. 5b) was beneficial for fast wound healing in this group and enhanced the formation of granulation tissue and epithelialization and wound closure [55]. ...
Article
The physiological healing process is disrupted in many cases using the current wound healing procedures, resulting in delayed wound healing. Hydrogel wound dressings provide a moist environment to enhance granulation tissue and epithelium formation in the wound area. However, exudate accumulation, bacterial proliferation, and reduced levels of growth factors are difficulties of hydrogel dressings. Here, we loaded platelet-rich fibrin-chitosan (CH-PRF) nanoparticles into the gelatin-chitosan hydrogel (Gel-CH/CH-PRF) by solvent mixing method. Our goal was to evaluate the characteristics of hydrogel dressings, sustained release of proteins from the hydrogel dressing containing PRF, and reduction in the risk of infection by the bacteria in the wound area. The Gel-CH/CH-PRF hydrogel showed excellent swelling behavior, good porosity, proper specific surface area, high absorption of wound exudates, and proper vapor permeability rate (2023 g/m ².day), which provided requisite moisture without dehydration around the wound area. Thermal behavior and the protein release from the hydrogels were investigated using simultaneous thermal analysis and the Bradford test, respectively. Most importantly, an excellent ability to control the release of proteins from the hydrogel dressings was observed. The high antimicrobial activity of hydrogel was confirmed using Gram-positive and Gram-negative bacteria. Due to the presence of chitosan in the hydrogels, the lowest scavenging capacity-50 value (5.82 μgmL⁻¹) and the highest DPPH radical scavenging activity (83 %) at a concentration 25 μgmL⁻¹ for Gel-CH/CH-PRF hydrogel were observed. Also, the hydrogels revealed excellent cell viability and proliferation. The wound healing process was studied using an in vivo model of the full-thickness wound. The wound closure was significantly higher on Gel-CH/CH-PRF hydrogel compared to the control group, indicating the highest epidermis thickness, and enhancing the formation of new granulation tissue. Our findings demonstrated that Gel-CH/CH-PRF hydrogel can provide an ideal wound dressing for accelerated wound healing.
... One decisive factor for the regenerative potential of PRF could be its capacity to release different growth factors. In addition to the activated platelets and leukocytes, the fibrin network acts as a reservoir for growth factors, enabling a continuous release profile [9,[16][17][18][19]. Particularly, vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF), and transforming growth factor-β1 (TGFβ-1) are considered promoters of wound healing and can be detected in the fibrin network [20]. ...
... Therefore, this blood-derived product can be individually adjusted and prepared according to specific clinical requirements as both liquid and solid matrices of PRF. Additionally, if required, solid matrices can be manufactured to create membranes [19] that may be used in guided bone regeneration approaches within dental implant therapy [25]. Furthermore, cylindric solid matrices can be created as clots to fill extraction sockets to prevent patients from suffering from severe alveolar ridge resorption and alveolus osteitis [26]. ...
... The consistency of PRF may, however, influence the amount and kinetics of growth factor release. Growth factor release within PRF can be evaluated using ELISA assays [9,19,32,33]. Sandwich ELISA was used in this study. Growth factors are detected as they bind with the capture antibodies located on the 96-well plate, as well as with the detection antibodies added. ...
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Aim: The purpose of this study was to obtain data concerning growth factor release within liquid and solid platelet-rich fibrin (PRF) matrices and to estimate the amount of potential interindividual variations as a basis for further preclinical and clinical trials. Therefore, we aimed to determine possible differences in the release of growth factors between liquid and solid PRF. Materials and Methods: Blood samples obtained from four subjects were processed to both liquid and solid PRF matrices using a standard centrifugation protocol. Five growth factors (vascular endothelial growth factor, VEGF; epidermal growth factor, EGF; platelet-derived growth factor-BB, PDGF-BB; transforming growth factor-β1, TGF-β1; and matrix metallopeptidase 9, MMP-9) have been evaluated at six time points by ELISA over a total observation period of 10 days (1 h, 7 h, 1 d, 2 d, 7 d, and 10 d). Results: Growth factor release could be measured in all samples at each time point. Comparing liquid and solid PRF matrices, no significant differences were detected (p > 0.05). The mean release of VEGF, TGFβ-1, PDGF-BB, and MMP-9 raised to a peak at time point five (day 7) in both liquid and solid PRF matrices. VEGF release was lower in liquid PRF than in solid PRF, whereas those of PDGF-BB and MMP-9 were higher in liquid PRF than in solid PRF at all time points. EGF had its peak release already at time point two after 7 h in liquid and solid matrices (hour 7 EGF solid: mean = 180 pg/mL, SD = 81; EGF liquid: mean = 218 pg/mL, SD = 64), declined rapidly until day 2, and had a second slight peak on day 7 in both groups (day 7 EGF solid: mean = 182 pg/mL, SD = 189; EGF liquid: mean = 81 pg/mL, SD = 70). Conclusions: This study detected growth factor release within liquid and solid PRF matrices with little variations. Further preclinical trials are needed to precisely analyze the growth factor release in larger samples and to better understand their effects on wound healing in different clinical indications.
... Additionally, there was a confusion in the literature concerning the reported parameters and the preparation methods [69,70]. Recent studies explored the role of the centrifugation process in the preparation of PRF [67,[71][72][73][74][75][76][77]. These studies have shown that the applied RCF has a crucial influence on the components and the bioactivity of PRF, thus influencing its therapeutic efficacy [67,[71][72][73][74][75][76][77]. ...
... Recent studies explored the role of the centrifugation process in the preparation of PRF [67,[71][72][73][74][75][76][77]. These studies have shown that the applied RCF has a crucial influence on the components and the bioactivity of PRF, thus influencing its therapeutic efficacy [67,[71][72][73][74][75][76][77]. Thereby, the application of a high RCF during the centrifugation of PRF results in a significantly lower number of platelets, leukocytes and growth factor concentrations compared to PRF-matrices that are prepared using a low RCF [67,[71][72][73][74][75][76][77]. ...
... These studies have shown that the applied RCF has a crucial influence on the components and the bioactivity of PRF, thus influencing its therapeutic efficacy [67,[71][72][73][74][75][76][77]. Thereby, the application of a high RCF during the centrifugation of PRF results in a significantly lower number of platelets, leukocytes and growth factor concentrations compared to PRF-matrices that are prepared using a low RCF [67,[71][72][73][74][75][76][77]. This phenomenon was proved in many studies and defined as the low-speed centrifugation concept (LSCC), which explained for the first time the role of the applied RCF in the preparation of blood concentrates [67]. ...
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Purpose To address the focused question: in patients with freshly extracted teeth, what is the efficacy of platelet-rich fibrin (PRF) in the prevention of pain and the regeneration of soft tissue and bone compared to the respective control without PRF treatment? Methods After an electronic data search in PubMed database, the Web of Knowledge of Thomson Reuters and hand search in the relevant journals, a total of 20 randomized and/or controlled studies were included. Results 66.6% of the studies showed that PRF significantly reduced the postoperative pain, especially in the first 1–3 days after tooth extraction. Soft tissue healing was significantly improved in the group of PRF compared to the spontaneous wound healing after 1 week (75% of the evaluated studies). Dimensional bone loss was significantly lower in the PRF group compared to the spontaneous wound healing after 8–15 weeks but not after 6 months. Socket fill was in 85% of the studies significantly higher in the PRF group compared to the spontaneous wound healing. Conclusions Based on the analyzed studies, PRF is most effective in the early healing period of 2–3 months after tooth extraction. A longer healing period may not provide any benefits. The currently available data do not allow any statement regarding the long-term implant success in sockets treated with PRF or its combination with biomaterials. Due to the heterogeneity of the evaluated data no meta-analysis was performed.
... [9] In vivo pre-clinical investigations showed the role of the clot structure in the vascularization and regeneration processes. [10] Comparative histological analysis demonstrated that, porous structure of A-PRF significantly facilitated the cellular penetration into the fibrin scaffold, the low-speed centrifugation concept and enhancing the growth factor release. [11] Metformin (MF), 1, 1-dimethylbiguanide, is a second class biguanide, used to manage type II diabetes mellitus. [12] The MF has the osteogenic effect through two mechanisms of action. ...
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Objective: This trial preformed to evaluate clinically and radiographically, the efficacy of growth factors producing/inducing materials; Advanced-Platelet-Rich Fibrin (A-PRF) and Metformin (MF) respectively, in the surgical treatment of intrabony periodontal defects. Methods: Forty-eight systemically healthy chronic periodontitis patients with intrabony defect were divided equally into 4 groups. First Group, patients treated by open flap debridement (OFD). Second Group, patients treated by OFD + 1% MF gel. Third Group, patients treated by A-PRF, which inserted into the intra bony defect (IBD). Fourth Group treated by (1% MF + A-PRF). Parameters were gathered at baseline 6 and 9 months. Results: The reduction in probing depth (PD) and clinical attachment level (CAL) was greater in the A-PRF + 1% MF patients group than other groups. Combination of A-PRF + 1% MF showed statistically significant reduction of IBD greater than all other groups. Conclusions: Usage of combination of A-PRF + 1% MF seems to be superior in gaining bone than surgical treatment by OFD, OFD + A-PRF or OFD + MF only.
... An in vitro study showed the absence of three key growth factors, namely VEGF, TGF-B1 and EGF, in the solid PRF-exudate compared to 10 days of sustained release of these in the liquid-PRF matrix. The VEGF and TGF-B1 showed a constant release while EGF peaked during the first hour with a progressive decrease up to day 10 (57). ...
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Objective: The study aims to identify the latest treatment protocols implementing platelet concentrates (PCs) to treat temporomandibular joint internal derangement (TMJ-ID) alone or in combination with arthroscopy/arthrocentesis.Background: Biosupplementation to treat TMJ-ID refers to the injection of an autologous product which possesses biological activity in the synovial joint, insofar as modulation of the inflammatory process, and can set the stage for tissue repair and/or tissue regeneration. PCs are increasingly used and tested, as an easily accessible biosupplement, for TMJ-ID. Methods: A systematic approach was used to perform the literature search. The study involves a presentation of recent data and a general discussion in the form of a narrative review regarding the biological characteristics and preparation protocols of PCs, the treatment protocols for TMJ-ID and clinical outcomes. Conclusions: The PCs described in the literature to treat TMJ-ID are platelet rich plasma (PRP), plasma rich in growth factors (PRGF), platelet-rich fibrin (PRF) and concentrated growth factors (CGF). All studies reported PCs as a beneficial therapeutic for TMJ-ID, in terms of arthralgia, joint noises and mandibular range of motion. Despite the biological differences among the various PCs, there exists similarities that could explain the reported benefits. The authors recommend clinicians the implementation of the acronym AR2T3 to standardize the reporting of PC preparations in future studies. Our findings suggest that non-surgical treatment through biosupplementation using PCs provides an alternative therapy or adjunct for painful TMJ-ID. The reduction in pain and dysfunction may avoid the need for TMJ surgery.
... The resulting PRF is composed of platelets, leukocytes, and their subgroups embedded in a fibrin scaffold [14]. Many studies outlined the regenerative capacity of PRF by releasing different growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and others [15][16][17]. Recently, the low speed centrifugation concept (LSCC) was introduced to optimize the regenerative capacity of blood concentrates by influencing the applied relative centrifugal force (RCF) [18]. ...
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Introduction: Resorbable synthetic scaffolds are promising for different indications, especially in the context of bone regeneration. However, they require additional biological components to enhance their osteogenic potential. In addition to different cell types, autologous blood-derived matrices offer many advantages to enhance the regenerative capacity of biomaterials. The present study aimed to analyze whether biologization of a PCL-mesh coated using differently centrifuged Platelet rich fibrin (PRF) matrices will have a positive influence on primary human osteoblasts activity in vitro. A polymeric resorbable scaffold (Osteomesh, OsteoporeTM (OP), Singapore) was combined with differently centrifuged PRF matrices to evaluate the additional influence of this biologization concept on bone regeneration in vitro. Peripheral blood of three healthy donors was used to gain PRF matrices centrifuged either at High (710× g, 8 min) or Low (44× g, 8 min) relative centrifugal force (RCF) according to the low speed centrifugation concept (LSCC). OP-PRF constructs were cultured with pOBs. POBs cultured on the uncoated OP served as a control. After three and seven days of cultivation, cell culture supernatants were collected to analyze the pOBs activity by determining the concentrations of VEGF, TGF-β1, PDGF, OPG, IL-8, and ALP- activity. Immunofluorescence staining was used to evaluate the Osteopontin expression of pOBs. After three days, the group of OP+PRFLow+pOBs showed significantly higher expression of IL-8, TGF-ß1, PDGF, and VEGF compared to the group of OP+PRFHigh+pOBs and OP+pOBs. Similar results were observed on day 7. Moreover, OP+PRFLow+pOBs exhibited significantly higher activity of ALP compared to OP+PRFHigh+pOBs and OP+pOBs. Immunofluorescence staining showed a higher number of pOBs adherent to OP+PRFLow+pOBs compared to the groups OP+PRFHigh+pOBs and OP+pOBs. To the best of our knowledge, this study is the first to investigate the osteoblasts activity when cultured on a PRF-coated PCL-mesh in vitro. The presented results suggest that PRFLow centrifuged according to LSCC exhibits autologous blood cells and growth factors, seem to have a significant effect on osteogenesis. Thereby, the combination of OP with PRFLow showed promising results to support bone regeneration. Further in vivo studies are required to verify the results and carry out potential results for clinical translation.
... К сожалению, покрытие трансплантатов адгезивными белками (ВКМ, плазмой или фибронектином) помимо улучшения адгезии ЭК способствует адгезии и агрегации тромбоцитов и повышению риска тромбообразования. В сравнении с цельным ВКМ и желатином фибрин демонстрирует меньшую активацию и адгезию тромбоцитов на поверхности, и соответственно, меньшую тромбогенность [64]. ...
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This review looks at the use of fibrin in vascular tissue engineering (VTE). Autologous fibrin is one of the most affordable biopolymers because it can be obtained from peripheral blood by simple techniques. A description and comparative analysis of the methods and approaches for producing fibrin gel is provided. The ability of fibrin to promote cell attachment and migration, survival and angiogenesis, to accumulate growth factors and release them in a controlled manner, are unique and extremely useful in VTE. Fibrin gels can serve as a three-dimensional matrix molded in different sizes and shapes to be applied in a variety of ways, including as a scaffold, coating, or impregnation material. Fibrin’s high porosity and biodegradability allows controllable release of growth factors, yet fibrinolysis must be tightly regulated to avoid side effects. We discuss the main methods of regulating the rate of fibrinolysis, as well as possible side effects of such exposure. Low mechanical strength is the main limitation in using fibrin as a scaffold for vascular tissue engineering. Possible options for increasing the strength properties of fibrin matrix and evaluating their effectiveness are presented. We propose that unique biocompatibility and ideal biodegradation profile of fibrin justify its use as a scaffold material for developing an ideal fully autologous small-diameter tissue-engineered vascular graft.
... In the last years, different procedures have been established to encourage bone and soft-tissue regeneration [14]. The advance of bioactive surgical extracts to reduce inflammation and increase healing procedure. ...
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Oroantral fistula is a pathological communication between oral cavity and maxillary antrum; commonly occurring after extraction of maxillary posterior teeth. Occasionally soft tissue closure is used in treatment of this defect but sometimes the soft tissue closure alone is not enough, so using of adjuvant material like Platelet Rich Fibrin is indicated. It is an autologous biomaterial; platelets and leukocytes of the blood in a strong natural fibrin matrix construct a complex structural design with excellent healing index. In this case report: after flap retraction and removing of the fistulas epithelial ling, Platelet rich fibrin used with soft tissue closure, then the follow up after 7-10-30 days show closure of the defect and cessation of symptoms after 10 days.Conclusions: Preparation technique of this biomaterial is simple and low-cost procedure with high soft tissue healing capability appears to be a very favorable option in treatment of oroantral fistula.
... Standardized iImmunohistochemical staining was performed according to standardized methods as previously described [7,23]. After deparaffinization and rehydration, the antigen was retrieved by the heat-induced epitope retrieval (HIER) This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. ...
Article
Blutkonzentrate, insbesondere das plättchenreiche Fibrin, („platelet-rich fibrin“, PRF), gewinnen zunehmend an Bedeutung in der regenerativen Medizin und Zahnmedizin. Mittlerweile sind unterschiedliche Blutkonzentratsysteme für die klinische Anwendung verfügbar. Deshalb besteht Bedarf an einem vertieften Verständnis dieser Systeme und der Möglichkeit ihrer Anwendung in unterschiedlichen klinischen Indikationen. Mit dem „low-speed centrifugation concept“ (LSCC) wurden erstmalig standardisierte und definierte Zentrifugationsprotokolle vorgestellt, um den klinischen Erfolg reproduzieren zu können. Dabei war es möglich, durch die Reduzierung der angewendeten Zentrifugalkraft eine signifikante Anreicherung der Blutkonzentrate mit Thrombozyten, Leukozyten und ihren Wachstumsfaktoren zu erreichen. Der vorliegende Beitrag beschäftigt sich mit der Zusammensetzung und Bioaktivität unterschiedlicher Blutkonzentrate und geht auf ihre Herstellung und den klinischen Einsatz ausführlich ein.
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Platelet-rich fibrin (PRF) is generated from the patients’ own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors’ concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.
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Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60–966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-β1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-β1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system. Graphical abstract
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Objectives: Research across many fields of medicine now points towards the clinical advantages of combining regenerative procedures with platelet-rich fibrin (PRF). This systematic review aimed to gather the extensive number of articles published to date on PRF in the dental field to better understand the clinical procedures where PRF may be utilized to enhance tissue/bone formation. Materials and methods: Manuscripts were searched systematically until May 2016 and separated into the following categories: intrabony and furcation defect regeneration, extraction socket management, sinus lifting procedures, gingival recession treatment, and guided bone regeneration (GBR) including horizontal/vertical bone augmentation procedures. Only human randomized clinical trials were included for assessment. Results: In total, 35 articles were selected and divided accordingly (kappa = 0.94). Overall, the use of PRF has been most investigated in periodontology for the treatment of periodontal intrabony defects and gingival recessions where the majority of studies have demonstrated favorable results in soft tissue management and repair. Little to no randomized clinical trials were found for extraction socket management although PRF has been shown to significantly decrease by tenfold dry sockets of third molars. Very little to no data was available directly investigating the effects of PRF on new bone formation in GBR, horizontal/vertical bone augmentation procedures, treatment of peri-implantitis, and sinus lifting procedures. Conclusions: Much investigation now supports the use of PRF for periodontal and soft tissue repair. Despite this, there remains a lack of well-conducted studies demonstrating convincingly the role of PRF during hard tissue bone regeneration. Future human randomized clinical studies evaluating the use of PRF on bone formation thus remain necessary. Clinical relevance: PRF was shown to improve soft tissue generation and limit dimensional changes post-extraction, with little available data to date supporting its use in GBR.
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Purpose The present study evaluated the platelet distribution pattern and growth factor release (VEGF, TGF-β1 and EGF) within three PRF (platelet-rich-fibrin) matrices (PRF, A-PRF and A-PRF+) that were prepared using different relative centrifugation forces (RCF) and centrifugation times. Materials and methods immunohistochemistry was conducted to assess the platelet distribution pattern within three PRF matrices. The growth factor release was measured over 10 days using ELISA. Results The VEGF protein content showed the highest release on day 7; A-PRF+ showed a significantly higher rate than A-PRF and PRF. The accumulated release on day 10 was significantly higher in A-PRF+ compared with A-PRF and PRF. TGF-β1 release in A-PRF and A-PRF+ showed significantly higher values on days 7 and 10 compared with PRF. EGF release revealed a maximum at 24 h in all groups. Toward the end of the study, A-PRF+ demonstrated significantly higher EGF release than PRF. The accumulated growth factor releases of TGF-β1 and EGF on day 10 were significantly higher in A-PRF+ and A-PRF than in PRF. Moreover, platelets were located homogenously throughout the matrix in the A-PRF and A-PRF+ groups, whereas platelets in PRF were primarily observed within the lower portion. Discussion the present results show an increase growthfactor release by decreased RCF. However, further studies must be conducted to examine the extent to which enhancing the amount and the rate of released growth factors influence wound healing and biomaterial-based tissue regeneration. Conclusion These outcomes accentuate the fact that with a reduction of RCF according to the previously LSCC (described low speed centrifugation concept), growth factor release can be increased in leukocytes and platelets within the solid PRF matrices.
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PurposeThe aim of this study was to analyze systematically the influence of the relative centrifugation force (RCF) on leukocytes, platelets and growth factor release within fluid platelet-rich fibrin matrices (PRF). Materials and methodsSystematically using peripheral blood from six healthy volunteers, the RCF was reduced four times for each of the three experimental protocols (I–III) within the spectrum (710–44 g), while maintaining a constant centrifugation time. Flow cytometry was applied to determine the platelets and leukocyte number. The growth factor concentration was quantified 1 and 24 h after clotting using ELISA. ResultsReducing RCF in accordance with protocol-II (177 g) led to a significantly higher platelets and leukocytes numbers compared to protocol-I (710 g). Protocol-III (44 g) showed a highly significant increase of leukocytes and platelets number in comparison to -I and -II. The growth factors’ concentration of VEGF and TGF-β1 was significantly higher in protocol-II compared to -I, whereas protocol-III exhibited significantly higher growth factor concentration compared to protocols-I and -II. These findings were observed among 1 and 24 h after clotting, as well as the accumulated growth factor concentration over 24 h. DiscussionBased on the results, it has been demonstrated that it is possible to enrich PRF-based fluid matrices with leukocytes, platelets and growth factors by means of a single alteration of the centrifugation settings within the clinical routine. Conclusions We postulate that the so-called low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRF-based matrices. Further studies are needed to evaluate the effect of cell and growth factor enrichment on wound healing and tissue regeneration while comparing blood concentrates gained by high and low RCF.
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Background: Demand for shorter treatment time is common in orthodontic patients. Periodontally Accelerated Osteogenic Orthodontics (PAOO) is a somewhat new surgical procedure which allows faster tooth movement via combining orthodontic forces with corticotomy and grafting of alveolar bone plates. Leukocyte and Platelet-Rich Fibrin (L-PRF) possess hard- and soft-tissue healing properties. Further, evidence of pain-inhibitory and anti-inflammatory potential is growing. Therefore, this study explores the feasibility, intra- and post-operative effects of using L-PRF in PAOO in terms of post-operative pain, inflammation, infection and post-orthodontic stability. Material and methods: A pilot prospective observational study involving a cohort of 11 patients was carried out. A Wilcko's modified PAOO technique with L-PRF (incorporated into the graft and as covering membrane) was performed with informed consent. Post-surgical pain, inflammation and infection were recorded for 10 days post-operatively, while the overall orthodontic treatment and post-treatment stability were followed up to 2 years. Results: Accelerated wound healing with no signs of infection or adverse reactions was evident. Post-surgical pain was either "mild" (45.5%) or "moderate" (54.5%). Immediate post-surgical inflammation was either "mild" (89.9%) or "moderate" (9.1%). Resolution began on day 4 where most patients experienced either "mild" or no inflammation (72.7% and 9.1%, respectively). Complete resolution was achieved in all patients by day 8. The average orthodontic treatment time was 9.3 months. All cases were deemed stable for 2 years. Conclusions: L-PRF is simple and safe to use in PAOO. Combination with traditional bone grafts potentially accelerates wound healing and reduces post-surgical pain, inflammation, infection without interfering with tooth movement and/or post-orthodontic stability, over a 2 years period; thus alleviating the need for analgesics and anti-inflammatory medications. Key words: Periodontally accelerated osteogenic orthodontics, leukocyte and platelet-rich fibrin, corticotomy, osteogenesis, grafts.
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Chronic wounds such as diabetic ulcers pose a significant challenge as a number of underlying deficiencies prevent natural healing. In pursuit of a regenerative wound therapy, we developed a heparin-based coacervate delivery system that provides controlled release of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) within the wound bed. In the current study, we used a polygenic type 2 diabetic mouse model to evaluate the capacity of HB-EGF coacervate to overcome the deficiencies of diabetic wound healing. In full-thickness excisional wounds on NONcNZO10 diabetic mice, HB-EGF coacervate enhanced the proliferation and migration of epidermal keratinocytes, leading to accelerated epithelialization. Furthermore, increased collagen deposition within the wound bed led to faster wound contraction and greater wound vascularization. Additionally, in vitro assays demonstrated that HB-EGF released from the coacervate successfully increased migration of diabetic human keratinocytes. The multifunctional role of HB-EGF in the healing process and its enhanced efficacy when delivered by the coacervate make it a promising therapy for diabetic wounds. This article is protected by copyright. All rights reserved. © 2015 by the Wound Healing Society.
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Abstract Choukroun´s PRF (platelet- rich fibrin) is obtained from blood without adding anticoagulants. In this study, protocols for standard platelet-rich fibrin (S-PRF) (rpm 2700, 12 minutes), and A (advanced)-PRF (rpm 1500, 14 minutes) were compared to establish by histological cell detection and histomorphometrical measurement of cell distribution the effects of the centrifugal force (speed and time) on the distribution of cells relevant for wound healing and tissue regeneration. Immunohistochemistry for monocytes, T and B -lymphocytes, neutrophilic granulocytes, CD34-positive stem cells and platelets was performed on clots produced from four different human donors.Platelets were detected throughout the clot in both groups, although in the A-PRF group more platelets were found in the distal part away from the buffy coat. T- and B-lymphocytes, stem cells and monocytes were detected in the surroundings of the buffy coat (BC) in both groups. Decreasing the rpm whilst increasing the centrifugation time in the A-PRF group gave an enhanced presence of neutrophilic granulocytes in the distal part of the clot. In the S-PRF group neutrophils were mostly found at the red blood cell (RBC) - BC interface.Neutrophilic granulocytes contribute to monocyte differentiation into macrophages. Accordingly, a higher presence of these cells might be able to influence the differentiation of host macrophages and macrophages within the clot after implantation .Thus, A-PRF might influence bone- and soft tissue regeneration, especially through the presence of monocytes/macrophages and their growth factors. The relevance and feasibility of this tissue-engineering concept have to be proven through in vivo studies.
Article
Aim: To investigate the influence of the use L-PRF as a socket filling material and its ridge preservation properties. Materials and methods: Twenty two patients in need of single bilateral and closely symmetrical tooth extractions in the maxilla or mandible were included in a split-mouth RCT. Treatments were randomly assigned (L-PRF socket filling vs. natural healing). CBCT scans were obtained after tooth extraction and 3 months. Scans were evaluated by superimposition using the original DICOM data. Mean ridge width differences between timepoints were measured at 3 levels below the crest on both the buccal and lingual sides (crest -1mm (primary outcome variable), -3mm and -5mm). Results: Mean vertical height changes at the buccal were -1,5 mm (± 1,3) for control sites and 0,5 mm (± 2,3) for test sites (p<0.005). At the buccal side, control sites values were respectively, -2,1 (±2,5), -0,3mm (±0,3) (p<0,005) and -0,1mm (±0,0), test sites values were respectively -0,6mm (±2,2) (p<0,005), -0,1mm (±0,3) and 0,0mm (±0,1) Significant differences (p<0,005) were found for total width reduction between test (-22,84%) and control sites (-51,92%) at 1mm below crest level. Significant differences were found for socket fill (visible mineralised bone) between test (94,7%) and control sites (63,3%). Conclusion: The use of L-PRF as a socket filling material in order to achieve preservation of horizontal and vertical ridge dimension at 3 months after tooth extraction is beneficial. This article is protected by copyright. All rights reserved.
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In the present study, the structure of two allogeneic and three xenogeneic bone blocks, which find application in dental and orthopedic surgery were histologically analyzed. The ultimate goal was to assess whether the components postulated by the manufacturer can be identified after applying conventional histological and histochemical staining techniques. Three samples of each material, i.e. allogeneic material-1 and -2 as well as xenogeneic material-1, -2 and -3 were obtained commercially. After decalcification and standardized embedding processes, conventional histological staining was performed in order to detect inorganic matrix, cellular or organic matrix components. Allogeneic material-1 showed trabecular bone-like structures, which were free of cellular components as well as of organic matrix. The allogeneic material-2 showed trabecular bone structures, in which connective tissue and cellular remnants were embedded. Additionally, some connective tissue, which resembled fat-like tissue, was found within this material. The xenogeneic material-1 showed trabecular bone-like structures and contained organic components comparable to that demonstrated for the allogeneic material-2. The xenogeneic material-2 showed trabecular bone structures with single cells located in lacunae. The xenogeneic material-3 also showed trabecular structures. Neither cellular nor organic matrix components were found within this material. According to the data of the present study, the allogeneic material-1 and the xenogeneic material-3 were the only investigated materials for which the obtained histological data were in accordance with the manufactureŕs advertised information. The remaining three materials showed discrepancies - although the manufacturers of all five bone substitute materials stated that their blocks were free of organic / cellular remnants. These data are of great clinical and material science interest. It seems that even patented processing techniques, are not always able to deliver reproducible materials. Although all manufacturers of all 5 bone blocks stated that their blocks were free of organic / cellular remnants, our histological analysis revealed that 3 out of 5 bone blocks did contain organic/cellular remnants. Such specimens might be able to induce an immune response within the recipient.