ORAL AND MAXILLOFACIAL SURGERY

85 No.6 June 1998
ORAL AND MAXILLOFACIALSURGERY
Editor: ktrry J. Peterson
Platelet-rich plasma
Growth factor enhancementfor bone grafts
Robert E. Marx, DDS,UEric R. Carlson,DMD,D Ralph M. Eichstaedt,DDS,CStevenR.
Schimmele.DDS.dJamesE. Strauss,DMD.e and KarenR. Georgeff.RN.r Miami, Fla.
UNIVERSITYOF MIAMI SCHOOLOF MEDICINE
Platelet-richplasmais an autologoussourceof plateletderivedgrowth factorand transforminggrowth factor beta that is
obtainedby sequestering
and concentratingplateletsby gradientdensitycentrifugation.
Thistechniqueproduceda concentrationof
human plateletsof 338% and identifiedplateletderivedgrowth factorand transforminggrowth factorbetawithin them.
Monoclonalantibodyassessment
of cancellouscellular marrow graftsdemonstrated
cells that were capableof respondingto the
growth factorsby bearingcell membranereceptors.The additionalamountsof thesegrowth factorsobtainedby addingplateletrich plasmato grattsevidenceda radiographicmaturationrale 1.62 to 2.16 timesthat of graftswithout platelet-richplasma.As
assessed
by histomorphometry,
there was also a greaterbone densityin graftsin which platelet-richplasmawas added(74.0'h +
'l
1%) than in graftsin which platelet-richplasmawas not added (55.1oh+ B'/.; p = 0.005).(Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 1998;85:638-46)
ln 1994Tayapongsaket al.l introducedthe novel idea of
adding autologoushbrin adhesive(AFA) to cancellous
bone during mandibularcontinuity reconstructions.They
identified earlier radiographicbone consolidationin 33
cases;they attributed this to enhancedosteoconduction
affordedto the osteocompetent
cells in the graft by virtue
of the fibrin network developedby AFA. They also
reported the remarkableadhesiveadvantageof binding
cancellousmiurow particles during graft placement.
Tayapongsaket al.l producedtheirAFA in a blood laboratory setting,separatingone unit of whole blood into the
red blood cell componentand the plasmafraction for use
aProfessorof Surgeryand Chief, Division of Oral and Maxillofacial
Surgery.
oAssociateProfessorof Surgery,Division of Oral and Maxillofacial
Surgery.
cStaff Oral and Maxillofacial Surgeon, VeteransAffairs Medical
Center, Dayton, Ohio; formerly, Fellow, Tumor and Reconstructive
Surgery, University of Miami School of Medicine.
dAssistant Professor of Surgery, Department of Oral Surgery, Oral
Medicine, Oral Pathology,Indiana University School of Dentistry;
formerly, Fellow, Tumor and Reconstructive Surgery, University of
Miami School of Medicine.
eChief Resident, Oral and Maxillofacial Surgery.
tharge Nurse, Oral and Maxillofacial Surgery, Health South
Doctors' Hospital, Coral Gables, Fla.
Receivedfor publicationOct. I, 1997;returnedfor revisionDec. 17,
1997; acceptedfor publication Jan. 8, 1998.
Copyright @ 1998 by Mosby, Inc.
1079-2l04l98l$5.M + 0 711U89479
638
over the following 2 to 3 weeksas a cryoprecipitate.This
was then thawed over a 24-hour period to yield a final
"frbrinogen-rich
concentrate"of l0 to 15 ml.
Since the early 1990swe have been exploring the
parallel but more specific sequestration and
concentrationof autologousplateletsin plasma(plateletrich plasma [PRP]) and studying the growth factors
containedwithin plateletsin relation to their biologic
enhancementof continuity bone grafts to the mandible.
The first purpose of this article is to introduce our
studiesof PRP; we presentdata documenting that PRP
increasesplateletconcentrationwhen placed into grafts,
showing the presenceof at least three growth factors
(platelet-derived growth factor [PDGF], transforming
growth factor beta I [TGF-bt] and transforming growth
factor beta 2 [TGF-b2]), and indicating that cancellous
milrow cells have receptors for these growth factors.
The secondpurposeof the article is to explore the potential of PRP to increasethe rate of bone formation in a
graft and enhancethe density of the bone formed at
6 months. The third purpose of the article is to
present a model of bone graft bone regeneration
illustrating the mechanismby which PRP may enhance
bone regenerationboth in rate and amount.
MATERIAT AND METHODS
Eighty-eight electivecancellouscellular marrow bone
graft reconstructionsof mandibularcontinuity defects5
E R A LP A T H O L O C Y
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Fig. l. TGF-b,. monoclonalantibodystainingshowscancellous marrow grafts to possesscells bearing receptorsfor
TGF-br growth factors; they are more concentratedaround
TGFvessels(perivascularloci) and on endostealosteoblasts.
b RI (V-22) epitome correspondto amino acids 158-179
(SantaCruz Biotechnologystains,original magnification
cm or greaterarisingfrom benign and malignanttumor
extirpationswithout radiotherapywere randomizedinto
two groups.One group receivedcancellouscellular
marrow grafts without added PRP.The secondgroup
receivedgraftswith PRP addedduring the bone-milling
phaseof graft preparationand applied topically atter
boneplacementinto the defect.For eachgraft the posterior ilium was usedas a donor site.
The PRP was obtained by means of an Electro
Medics 500 gradient density cell separator
(Medtronics)used in the operatingroom simultaneously with bone graft harvesting.This cell separator
withdraws 400 to 450 ml of autologouswhole blood
through a central venous catheter placed during
surgery.With a centrifugespeedof 5600 RPM, whole
blood is drawn at a rate of 50 ml/min. As it withdraws
the blood the separatoraddscitratephosphatedextrose
(CPD) at a ratio of I ml of CPD to 5 ml of blood to
achieveanticoagulation.The blood is then centrifuged
into its three basic components;red blood cells, PRP
(sometimesreferred to as "buffy coat"), and plateletpoor plasma (PPP). Becauseof differential densities,
the red blood cell layer forms at the lowest level, the
PRP layer in the middle, and the PPP layer at the top.
The cell separatorincrementallyseparateseach layer,
from the lessdenseto the more dense;thereforeit separatesPPP first (about 200 ml) and PRP second(about
70 ml), leaving the residualred blood cells (about 180
ml). Once the PPP is collected,the centrifugespeedis
lowered to 24OORPM to allow for a precise separation
of the PRP from the red blood cells. In fact, both our
experience and testing by Reeder et al.4 have shown
Marx et al. 639
that the plateletsmost recently synthesized,and therefore of greatestactivity, are larger and mix with the
upper I mm of red blood cells, so that this layer is
included in the PRP product.This impartsa red tint to
the PRP.which would otherwisebe straw colored.The
red blood cells and PPPare returnedto the patientfrom
their collection bags through either the central venous
catheteror a peripheralvenousaccess.
This procedure takes approximately 20 to 30
minutes.However,it is accomplishedsimultaneously
with either the bone harvestingprocedureor preparation of the recipient tissues,and thereforeit does not
add to operatingroom time. The Medtronicscell sepaof most operatingrooms
rator is in the armamentaria
that are also used for major orthopedicand cardiovascular surgery;there are thus no additional expenses
except those associatedwith disposablecatheters,a
central venousline, and an internalcentrifugebowl,
which togethercost approximately$300.00.
Samplesof PRP and venousblood were submittedfbr
machine platelet counts and a smear with Giemsa
stainingfor a manualcount.Two additionalPRP smears
were stainedwith monoclonalantibodystains(Santa
Cruz Biotechnology,Santa Cruz, Calif.). One was
stainedfor PDGF and the other for TGF-b. A sampleof
the autogenousgrafi material was placed in fbrmalin,
processedwith a slow formic acid decalcification,and
stainedwith monoclonalantibodiesto identify PDGF
receptors(PDGFT)and TGF-b receptors(TGF-b,).
The PRP application requires initiating the
coagulationprocesswith a mixture of l0 ml of l0%o
calcium chloride mixed with 10,000units of topical
bovine thrombin (Gentrac). The protocol for PRP
applicationrequiresthe use of an individual 1O-ml
syringe for each mix. Each mix draws, in order, 6 ml
of PRP, I ml of the calcium chloride/thrombinmix,
and I ml of air to act as a mixing bubble.The syringe
is agitatedfor 6 to 10 secondsto initiateclotting.The
PRP,now a gel, is addedto the graft in severalmixes.
If several mixes are used, a sterile new syringe is
requiredat each mix. The addition of a small amount
of calcium chloride and thrombin from a reused
syringe can coagulatethe remainderof the PRP in its
container.Once the PRP is addedto the graft the fibrin
formation binds the otherwise loose cancellous
cellular marrow together to assist the surgeon in
sculpting the graft. The fibrin network establishedin
the graft is thought to assist the osteoconduction
componentof bone regeneration.l
The bone grafts were allowed to consolidate and
mature for 6 months. Panoramic radiographs were
taken at the 2-.4-. and 6-month intervals. The unlaby two investigabeledpanoramicfilms were assessed
tors (S.R.S.and R.E.M.) as to the age of the graft at
640
O R A LS U R C E RO
YR A LM E D I C I N E
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Marx et al.
s
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Fig. 2. Normal plateletdensity in peripheralblood smear
(Giemsastain,originalmagnificationxl0).
Fig. 3. Obviousconcentrationof plateletsseenon PRP smear
indicates transplantationof additional platelets into graft.
Cytoplasmicgranules(alphagranules)are known location of
PGGF,TGF-b,, and TGF-br, among other growth factors.
Table l. Platelet counts: 3387o increase
BuseLineplatelet counl
232,000
( l l r.000-s23,000)
PRPplatelet count
785,000
(595,000-1,100,000)
each interval. The ratio of assessedgraft maturity to
actual graft maturity gave a numeric index of graft
maturity (graft maturity index IGMII). At least one
osseointegrated
implant was placed into each graft at
the 6-monthinterval.The placementof implant fixtures
throughuseof a /3i implant (Implant Innovations,Inc.)
with a diameterof 4.0 mm allowed a core bone specimen 2.9 mm in diameterto be processedfor histomorphometry and for monoclonalantibody staining fbr
PDGFr and TGF-br. Histomorphometry was accomplishedwith a semiautomatic
computerimage system
(SMI Unicomp,Atlanta, Ga.).This systemprojectsthe
histologic image onto a video screen.Random areas
were traced on a digitizing pad to calculate area of
mineralizedbone matrix versustotal areaof view. The
area of mineralized bone matrix was recorded as
trabecularbone area(TBA) versusmarrow spacearea.
For purposesof comparisonand control, l0 resection
specimensof the midbody of the mandible were
assessedwith the same histomorphometrictechnique,
and a TBA was calculatedfor each.
RESUTTS
PRPmonoclonalantibodystudy
The platelets sequesteredby the centrifugation
process showed an intense uptake of both PDGF and
TGF-b monoclonal antibodies in all slides, thus
confirming the presenceand retention of these growth
factorsin the PRP preparation.
Fig. 4. Functioningcancellousmarrow graft where PRP was
not usedis well consolidatedat 6 months.
Bone graft harvest material monoclonal
antibody study
All slides of harvestedcancellouscellular marrow
showed cell populations that tested positive for
receptorsto PDGF and TGF-b. It was observedthat
most of these cell populations were centered about
blood vesselsin a perivascularsheet.Lesser numbers
were observed on the trabeculae of the cancellous
bone's endosteal surface and randomly dispersed
betweenfat cells in the marrow.(Fig. l). Theseresults
identified the presence of marrow stem cells and
osteoprogenitorcells within human cancellous mzrrrow
capable of responding to the increased PDGF and
TGF-b in the PRP preparation.
Plateletcount study
Platelet counts done on each patient yielded a
mean plateletcount value of 232,000,with a range of
I I1,000 to 523,000. The PRP mean platelet
count was 785,000, with a range of 595,000 to
1,100,000. These values confirmed the platelet
O R A LS U R C E RO
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Marx et al.
641
Fig. 5. Functioningcancellousmarrow graft where PRP was
used shows an enhancedmaturity and bone consolidationat
6 months.
Fig. 7. Trabecularbone area of human posterior mandible.
Mean trabecularbone areais 38.97o+ 60/o.
Table ll. Graft maturity index
Time (m,)
GraJts
2
0.92
0.88
1.06
A
6
Graft + PRP
z.t6
1.88
1.62
0.001
0.001
0.001
Tablelll. Histomorphometricfindings at 6 months
Native mandible(10)
Bone grafts (44)
Bone grafts with PRP (214)
antibodystainingshowscell
Fig. 6. TGF-b,.monoclonal
producingTGF-b,presumably
to maintainboneformationin
graftasan autocrinestimulation.
TGF-bl.(v) epitomecorre(SantaCruz Biotechnology
spondsto aminoacids352-3'17
sequestrationability of the process and quantified the
concentrationas 338Voof baseline platelet counts
(TableI; Figs. 2 and 3).
Assessment
of radiographicgraft maturity
The results of the panoramic radiographic assessment are illustrated in Table ll. At 2 and 4 months the
grafts without PRP growth factor additions were
assessedat or just below their actual maturity; at 6
months they were assessedat orjust aheadof the actual
graft maturity. The grafts with PRP growth factor
additions were consistently assessedeither at or at
slightly more than twice their actual maturity, with
ratios of 2.16 at 2 months. 1.88 at 4 months. and 1.62
at 6 months. Each comparison of the average graft
maturity index values of PRP-addedgrafts with those
of the grafts without PRP was assessedby means of a
Student / test. The p value for each comparison was
0.001 (Figs. 4 and 5).
38.97ot 6Vo
! 8Vo
55.1o/o
7 4 . 0 9 ox . l l % a
0.005
0.005
with monoclonal
Six-monthgraftassessment
antibodies
Processedcore bone specimensof eachgraft type at 6
months demonstrateda continued production of TGF-b.
Monoclonal antibodiesidentified TGF-b but not PDGF
by marrow stem cells and endostealosteoblasts.The
TGF-b-positive cells were noted to be concentratedon
the trabecular bone endostealsurface,on the periosteal
surface,and within active marrow stem cells. Only rare
cells stainedpositive for PDGF and were thus interpreted as nonreactive(Fig. 6).
with histomorSix-monthgraftassessment
phometry
The results of the histomorphometric study, which
are illustrated in Table III, indicated that bone grafts in
general produce a trabecular bone area greater than
that of native posterior mandible (55.17o ! 8Vo vs
38.9Vox.67o;p = 0.005). This was not unexpectedand
has been reported earlier.2'3However, bone grafts with
growth factors added by means of PRP demonstrated
even greater trabecular bone density than did bone
642
Marx et al.
Fig. 8. Trabecularbone area of non-PRP-enhancedcancellous cellular marow graft at 6 months.Mean trabecularbone
area is 55.17o+ 8olo.Note that bone is partially woven bone
(immaturephaseI bone) and partially lamellar bone (mature
ORALMEDICINEORALPATHOLOGY
ORALSURGERY
June 1998
Fig. 9. Trabecular bone area of PRP-enhancedcancellous
marrow graft at 6 months. Mean trabecular bone area is
74.0Vo+ I l7o. Note that bone is all lamellar (phaseII) bone,
indicativeof advancedmaturity.
phaseII bone).
DtscussroN
grafts withoutPRP (74.07o + ll%o vs 55.lVo + 8Vo,P
Natureof PDGFandTGF-bgrowthfactors
= 0.005;Figs.7, 8, and 9).
The results in these studies suggestedthat PRP
addition acceleratedthe rate of bone formation and
the degree of bone formation in a bone graft through
at least the first 6 months. The data indicatedthat
increased numbers of platelets containing the
documented growth factors PDGF and TGF-b, as
well as other growth factors that have been identified
can be technically
in the alpha granulesof platelets,a-6
sequestered,concentrated,and added to bone grafts
along with the fibrin network originally identified
by Tayapongsaket al.l Cell separatortechnology
allows the specific sequestrationand harvesting of
only the platelet- and leukocyte-rich layer, avoiding
the concentratedbut mixed, and therefore diluted,
PRP and PPP combinedfractions of earlier studies.l'4
Furthermore these studies showed that cancellous
miurow grafts contain cells bearing PDGF and TGF-b
receptors as the probable targets of PRP, as
well as being intimately involved in the bone
regenerationprocess.These cells, referred to as stem
cells by Caplan,Twere found in three locations in
cancellous marrow. The perivascular location of
marrow stem cells has been suggested by many
and it accountedfor the greatestnumberof
authors,8-I0
cells with such receptors.The endosteal location
probably represents osteoblasts or preosteoblasts,
which are known to be activatedby PDGF and TGF-b.
The more finely dispersedinterstitial cells probably
represent diffuse stem cells occupying locations of
structural opportunity within the muurowspace.
PDG|. A glycoprotein, PDGF has a molecular weight
of approximately 30 kd. 12 Although it was first
describedin the alpha granulesof platelets,it is also
synthesizedand secretedby other cells, such as
It seemsto be the first
and endothelium.13
macrophages
growth factor present in a wound, and it initiates
connectivetissuehealing,including bone regeneration
and repair.In humans,it existsmostly as a heterodimer
of two chains-termed A and B chains---of about equal
size and molecularweight (approximately14 kd to l7
kd;.13't+In lesserquantities,A-A and B-B homodimers
exist in human beings with the same activity.l3The
reasonfor three distinct dimeric forms remains unclear,
but differentialbinding by various receptorcells, such
as endothelium, fibroblasts, macrophages,and mturow
It is known to
stem cells, has been suggested.l3'15
emerge from degranulating platelets at the time of
injury. Its mechanism is to activate cell membrane
receptors on target cells, which in turn are thought to
develop high-energy phosphatebonds on internal cytoplasmic signal proteins; the bonds then activate the
signal proteins to initiate a specific activity within the
target cell.15The most important specific activities of
PDGF include mitogenesis(increasein the cell populations of healing cells), angiogenesis(endothelial
mitoses into functioning capillaries), and macrophage
activation (debridementof the wound site and a secondphasesourceof growth factors for continued repair and
bone regeneration).There are approximately 0.06 ng of
PDGF per one million platelets,a fact that underscores
this molecule's great potency.l6 Stated in other
terms, there are 6 x 10-17g of PDGF, or about 1200
molecules of PDGF, in every individual platelet.16'17
ORALSURCERY
ORALMEDICINEORALPATHOLOCY
Volume85,Number6
0stcocytes
Oste,oblasts
Stem Cclls
Marx et al. 643
'
1t-
:
.
a
@
Po2:5-lo
Platletg (pocr)
z
(T6F-b)
Fibrin
L
a
%t
@
pl{'*-o
Qa
p:
--
(i
\7e
q{-;
Or Gradient=so'4o--h
lacr.ophageAttractant
\?
pH=,t.42
P O z =4 5 - 5 5 - - H g
Fig. 10. Basic cells, biochemistry,and growth factorsassociatedinside and outside wound spaceof cancellous marrow sraft.
Therefore a threefold or greater concentration of
platelets,as was measuredin PRP,can be expectedto
havea profoundeffect on wound healingenhancement
and bone regeneration.
TGF-b. The term tansforming growth factor beta is
appliedto the superfamilyof growth and differentiating
protein(BMP)
factorsof which the bonemorphogenetic
family, containingat least l3 describedBMPs, is a
member.l8The TGF-bs referredto and studiedin this
article are the TGF-bl and TGF-b, proteins,which are
the more proteanand generic growth factors involved
with generalconnectivetissuerepairand boneregeneration.5'6TGF-br and TGF-b, are proteinsthat havemolecular weights of approximately2-5kd.)'b Like PDGF,
they are synthesized and fbund in platelets and
as well as in someothercell types.When
macrophages,
or activelysecretedby
releasedby plateletdegranulation
macrophages,they act as paracrinegrowth factors (ie.
growth factors secretedby one cell exerting its effect on
an adjacentsecondcell), affecting mainly fibroblasts,
However,each
marrow stemcells, and the preosteoblasts.
of thesetargetcells has the ability to synthesizeand
secreteits own TGF-b proteinsto act on adjacentcellsin
a paracrinefashionor act on itself as an autocrinegrowth
factor (ie, a growth factor which is secretedby a cell and
actson its own cell membraneto continueits activity).19
TGF-bsthereforerepresenta mechanismfor sustaininga
long-termhealingand bone regenerationmodule and
evenevolveinto a boneremodelingfactorovertime.The
mostimportantfunctionsof TGF-b, andTGF-b, seemto
be the chemotaxisand mitogenesisof osteoblastprecursors,and theyalsohavethe ability to stimulateosteoblast
depositionof the collagenmatrix of wound healingand
of bone.20In addition,TGF-bsinhibit osteoclastformation and bone resorption,thus favoring bone formation
over resorptionby two dill'erentmechanisms.2l
A model of bone graft regeneration
From our previouswork and from a new knowledge
of growth t-actorinfluences,we can proposea reasonable model for the bone regenerationobserved in
cancellouscellular marrow grafts.This model can also
illustrateout wherePDGF andTGF-b growth factors,at
least, influence bone regenerationnormally, and how
increasedquantities of each factor through PRP
producedthe faster rate of bone formation and the
greaterquantity of bone seenin this study.
A cancellouscellular marrow graft, whether fbr a
mandibularcontinuitydefect,an alveolarcleft, or a sinus
lift surgery,is placedinto a dead spacefilled with clotted
blood.The wounddeadspaceis hypoxic(pOr,5-10mm
Hg) and acidotic(pH, a-6) and containsplatelets,leukocytes,red blood cells, and fibrin in a complex clot adjacent to the transferredosteocytes,endostealosteoblasts,
O R A LS U R C E R Y
Marx et aL
ORALPATHOLOCY
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Cell Aitosis
%--%
"--N./
ri\
Capillary
Buddilg
_lllgcnophages
12
(ADAF)
(r'rDGF)
(OAF)
(br6F)
( PD6F)
Fig. ll. By tlay -3capillary ingrowth beginsin responseto PDGF and TGF-b. Stem cells and endosteal
osteoblastsmitose in responsek) thesesamegrowth tactorsto createa cell populationcapableof producing
functionalquantityof new bone.Macrophagebecomesmain growth lactor elaboratingcell, inasmuchas
plateletshavecompletelydegranulatedby now.
(pig. l0). The marrowstem
anil marrowstemcells2'22'21
cells,
cells, which are the primary bone-regenerating
(approximately
I
normally exist in very small numbers
human
per 400,000 structuralcells in a 5O-year-old
Just outsidethe surgeon'speriosteallevel
being).7'10
The tissue
closure,the tissueis physiologicallynormal.23
pH
physiologic
is normoxic(pOr, 45-55 mm Hg) and at
cells,
structural
(pH,7 .42).and it containsa populationof
stemcells (alsoin very small numbers),
healing-capable
and cut capillarieswith clots and exposedendothelial
cells. This complex environment-simplified in our
model-is the productof millions of yearsof evolution;
it starts,maintains,and promotesmaturebone repair
relatedto injury and can be usedby surgeonstoday tcl
bonethroughbonegrafting.
regenerate
The initiation of bone regenerationstartswith the
releaseof PDGF and TGF-b from the degranulationc'f
plateletsin the graft.The PDGF stimulatesmitogenesis
transofthe marrowstemcellsandendostealosteoblasts
several
by
numbers
ferrecl in the graft to increasetheir
of
ordersof magnitude.It also beginsan angiogenesis
endothelial
inducing
capillarybuddinginto the graft by
cell mitosis.The TGF-b initially activatesfibroblastsand
to mitoseand increasetheir numbers,as
oreosteoblasts
well as promoting their differentiationtoward mature
ContinuedTGF-b secretion
functioningosteoblasts.
to lay down bone matrix and
influencesthe osteoblasts
the fibroblast to lay down collagen matrix to support
capillaryingrowth.Theseactivitiesbeginimmediatelyon
woundclosure.By thethird day capillariescanbe seento
penetrate
the graft.Completecapillarypermeationof the
g r a f i i ss e e nb y d a y 1 4 t o d a y l 7 r 1 F i g sI. I a n d l 2 ) .
This initial fluny of cellularactivity,thoughit is also
the resultof someothergrowth factors,is primarily the
direct result of PDGF and TGF-b. Its evolutionary
purposeis simply organismenergyefficiency.That is,
most body cells are differentiatedstructural or functional cells. It would be energy-fbolishand actually
impossiblefbr an organism'senergyeconomyto maintain a largepopulationof cells for healingand no other
purpose.Instead,mammalianevolutionproceededto
m a i n t a i no n l y m i n u t e l y s m a l l n u m b e r so f h e a l i n g capablestemcells (l/100,000in a teenager'l/250'000
in a person35 yearsof age, l/400,000 in a person50
years of age and l/1,200,000in a person 80 years of
age)10:growth factorsare relied on to rapidly increase
the numbersof thesecells and promotetheir activity
during a time of injury..
Marx et al.
tttf OnnL PATHOLOCY
ORALt.leOtCt
ORALSURCERY
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Volume85,Number
Circulating9[em&lls
f^o
u;\t-,r
StcmCell
Bonc
Fibrin
PD6F+
PHt7.4?.
-.t)
Release
l0r
|4DGF f
D
oF anp
cells are now sustainingthemselves
Fig. 12. By day 14 capillary ingrowth is nearly complete.Bone-forming
of wound by capillary perfunormalization
with their own autoc.ne pioauction of rGF-b. with physiologic
sion, macrophagefunction beginsto dissipate'
The life spanof a plateletin a wound and the period
of the direct influenceof its growth factorsis lessthan
5 days. The extensionof healing and bone regeneration activity is accomplishedby two mechanisms'The
first is the increase and activation of marrow stem
cells into osteoblasts,which secreteTGF-b themselves.The secondand more dominant mechanism
seems to be the chemotaxis and activation of
macrophagesthat replacethe plateletsas the primary
r;our"" of growth factors after the third day'20'24The
macrophage is attracted to the grafi by the actions of
PDGF and by an oxygen gradient between the graft
dead space and the adjacent normal tissue that is
greater than 20 mm Hg.23'25In fact, the graft's
inherent hypoxia of 5 to l0 mm Hg establishesan
oxygen gradient of 30 to 40 mm Hg adjacentto the
normoxic tissues,which are at 45 to 55 mm Hg'
As PDGF faclesin influence, macrophage-derived
growth and angiogenic factors take over' However'
macrophage-derivedgrowth factors and angiogenic
fhctors may actually be identical to PDGF-only
synthesized by macrophages instead of by
platelets.The marrow stemcells will secreteTGF-b to
continue a self-stimulation of bone formation as an
autocrine response;the identification of continued
TGF-b activity arising from marrow cells in our
study confirms this (Fig. 6). By 4 weeks, the
revascularizedgraft eliminatesthe oxygen gradient
neededto maintain macrophageactivity' Thus the
macrophageleavesthe area' no longer required by a
graft that is now self-sustainingeven though immature, with woven osteoid bone rather than mature
lamellar 6on".23'25
The actual maturationof the graft from a disorganized
woven bone into a mature lamellar bone with haversian
systemsinvolvesthe third and final growth factor group
in this model, which was not part of this study and is not
contained in PRP. It is bone morphogeneticprotein
(BMP). As bone matrix is formed and then mineralized
by osteoblasts,BMP is laid down within the bone
matrix.26This acid-insolubleprotein is then releasedby
the osteoclasticresorption of normal bone remodeling,
which progressesat a rate of 0.77o per day in normal
bone but may occur as rapidly as 57oto 8Voper day in a
maturing bone graft.2'27The releasedBMP links bone
resorptionto new bone formation by acting on adjacent
stemcells to increasetheir numbersand differentiateinto
functioning osteoblaststhat actively secretebone matrix'
Thus the graft cycle from a cellular transplantthat is
placed into a complex biochemical environment
progressesto a maturefunctional bone that is self-maintaining through the normal resorption-remodelingcycle'
I
i
I
II
Ir
646
Marx et al.
coNctusroNs
Today'sunderstanding
of bone sciencerecognizesthe
pivotal role of growth f'actorsin clinical bone grafting
success.
This articleelucidatesthe mechanismof action
and the pointsof influencethat the fundamentalgrowth
factors PDGF and TGF-b exert on bone regeneration.
The amplificationof PDGF andTGF-b throughthe technique of plateletsequestration
and concentrationinto a
platelet-richplasmais seenas an availableand practical
tool for enhancingthe rate of bone formation and the
final quantity of bone formed. The fact that PRP is an
autologouspreparationintroducedat the time of surgery
eliminatesconcernsabout diseasetransmissionand
immunogenicreactions,which are associated
with allogeneicor xenogeneicpreparation,and about the possibility of mislabelinga sample, which might occur
througha laboratorysystem.
In this study it was shownthat PRP indeedcontainsa
concentrationof plateletsand a concentrationof growth
f'actors.The graft material was shown to contain cells
responsiveto thesegrowth factors. Finally, our study
presentedevidencethat thesegrowth factor additionsto
bone grafts produced a quantifiably enhancedresult in
comparisonwith grafts performedwithout its use.
The authorswish to emphasizethat PDGF and TGF-b
are not the only growth factorsthat influenceboneregeneration and are not the only growth factors containedin
PRP.Although it is an admittedly oversimplified model
that focuseson the growth factors availableto surgeons,
the mechanismof action presentedin this article is a
good working model with which surgeonscan understandand plan strategiesfor growth factor uses.
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Reprtnt requests:
Robert E. Marx, DDS
Division of Oral and Maxillofacial Surgery
University of Miami School of Medicine
5000 University Drive
Coral Gables,FL 33146