Platelet-rich plasma influence on human ... - Clauberth Oliveira

Cimara Fortes Ferreira
Márcia Cristina Carriel
Gomes
José Scarso Filho
José Mauro Granjeiro
Cláudia Maria Oliveira
Simões
Ricardo de Souza Magini
Authors’ affiliations:
Cimara Fortes Ferreira, Department of Dental
Implantology, University of Santa Catarina,
Florianópolis, Brazil
Márcia Cristina Carriel Gomes, Department of
Biotechnology, University of Santa Catarina,
Florianópolis, Brazil
José Scarso Filho, Department of Oral Surgery,
University of Araraquara, Araraquara, Brazil
José Mauro Granjeiro, Department of Biochemistry,
School of Dentistry, Bauru, Brazil
Cláudia Maria Oliveira Simões, Department of
Pharmaceutical Sciences, University of Santa
Catarina, Florianópolis, Brazil
Ricardo de Souza Magini, Department of
Periodontology, University of Santa Catarina,
Florianópolis, Brazil
Correspondence:
Dr Ricardo de Souza Magini
Universidade Federal de Santa Catarina – Centro de
Ensino e Pesquisa em Implantes Dentários
(UFSC-CEPID)
Centro de Ciências da Saúde–Campus
Universitário Trindade
88040-970
Florianópolis, SC
Brazil
Tel.: þ 55 48 331 9077
Fax: þ 55 48 234 1788
e-mail: cimarafortes@hotmail.com
Date:
Accepted 18 October 2004
To cite this article:
Ferreira CF, Gomes MCC, Filho JS, Granjeiro JM,
Simões CMO, Magini RS. <strong>Platelet</strong>-<strong>rich</strong> <strong>plasma</strong>
<strong>influence</strong> on human osteoblasts growth.
Clin. Oral Impl. Res. 16, 2005; 456–460
doi: 10.1111/j.1600-0501.2005.01145.x
Copyright r Blackwell Munksgaard 2005
456
<strong>Platelet</strong>-<strong>rich</strong> <strong>plasma</strong> <strong>influence</strong> on
human osteoblasts growth
Key words: human osteoblasts; platelet-<strong>rich</strong> <strong>plasma</strong>; proliferation
Abstract
Objective: The <strong>influence</strong> of progressively high concentrations of platelet-<strong>rich</strong> <strong>plasma</strong> (PRP)
on human osteoblast hFOB1.19 proliferation was evaluated.
Material and methods: The PRP was obtained from a human source. Two experiments were
conducted. In the first one, PRP was diluted to 50%, 25%, 12.5% and 6.125% (v/v) with
culture medium (Modified Eagle’s Medium (MEM) : Ham’s F12 Medium (HAM-F12%) and
1% antibiotics–antimicotic) supplemented with 10% of fetal bovine serum (FBS). In the
second experiment, all conditions were identical except for the absence of FBS in the
culture medium.
Results: The results of the osteoblast proliferation test were higher when stimulated by the
50% PRP dilution, with or without FBS. A further study is suggested to determine if
concentrations above 50% could cause higher rates of osteoblast proliferation. In this study,
the results were not statistically different (Po0.05) with 12.5% and 6.125% PRP dilutions.
Additionally, it was shown that FBS is not necessary for PRP-mediated induction of
osteoblast proliferation.
Conclusion: This study concluded that PRP promotes osteoblast proliferation and
suggested its clinical application to bone graft procedures in implant dentistry.
Scientific studies have established platelet<strong>rich</strong>
<strong>plasma</strong> (PRP) as a therapeutic strategy
in orthopedics (Ganio et al. 1993), in implant
dentistry (Anitua 1999; Kassolis et al.
2000; Gruber et al. 2002), and as an aid for
bone graft compaction as well as for hemostasis.
PRP is a non-toxic, autogenous
material that does not cause immune reactions
when applied to the original donor.
PRP possesses the advantage of being immediately
available preoperatively, and
consequently histocompatible, and being
incapable of transmitting infectious diseases
(Whitman et al. 1997). PRP also
contains high amounts of mitogenic polypeptide
proteins, platelet-derived growth
factors (PDGFs), b-transforming growth
factors (TGF-b’s) and I-insulin-like growth
factors, which appear to accelerate the
osteogenesis in oral and maxillofacial surgery,
as well as platelets, which are a
source of growth factors that induce bone,
epithelial and connective tissue repair. It
has been successfully used since the 1970s.
Matras (1970) used <strong>plasma</strong> to form fibrin
‘glue’ preparations obtained from blood
centrifugation for skin graft procedures
in mice. Studies showed the beneficial
effects of PRP to repair cutaneous ulcers
(Knighton et al. 1990; Lynch 1991). PRP
promotes acceleration of surgical wound
repair by means of growth factors present
in the platelets, which are the universal
initiators of the healing process (Ganio

et al. 1993; Tayapongsak et al. 1994; Anitua
1999). Its benefits were also confirmed
in oral and maxillofacial surgeries, (Ganio
et al. 1993; Kassolis et al. 2000). Lucarelli
et al. (2003) investigated mesenchymal
stem cell proliferation in culture medium
supplemented with PRP and verified that
the use of 10% PRP was sufficient to
accelerate mineralization in vitro. Clinicians
and researchers have investigated
the use of PRP in dentistry as a form of
accelerating the natural healing process.
Carlson & Roach (2002) showed that PRP
and its growth factors are promising for
surgical wound healing. The use of PRP,
in association with autogenous bone grafts
in implant dentistry, was shown to optimize
the quality and quantity of newly
formed bone (Scarso Filho 2002).
A systematic review of maxillary sinus
augmentation procedures on the survival
of endosseous dental implants was done
when the efficacy of the sinus augmentation
procedure was compared with various
surgical grafting materials, including the
use of PRP associated with bone graft
material. In spite of the favorable responses
shown in the literature in using
PRP as an adjuvant for bone-grafting
procedures, the authors of this systematic
review concluded that there are insufficient
data to recommend the use of PRP
in sinus graft surgery (Wallace & Froum
2003).
Another study on the use of PRP associated
with autologous bone as filling
material for sinus lift procedures shows
increased bone maturation rate and improved
bone density when PRP, or its
recombinant growth factors, are added to
small bony defects or to larger defects that
use autogenous bone as the grafting material.
Histomorphometric analysis indicated
that the addition of PRP to the grafts did
not show significant difference either in
vital bone production or in interfacial
bone contact on implants placed in these
sites (Froum et al. 2002). Other studies
using animal models showed that PRP
associated with autogenous bone did not
enhance bone formation (Roldan et al.
2004; Schlegel et al. 2004; Wiltfang et al.
2004).
The effect of PRP on bone regeneration,
when associated to autogenous bone grafts
was evaluated in vitro in a canine model
(Choi et al. 2004). The findings suggested
that the addition of PRP does not appear to
enhance new bone formation.
The purpose of the present study was to
evaluate progressively high concentrations of
PRP on proliferation of human osteoblasts.
Material and methods
Cell culture
Human osteoblasts (hFOB1.19, no. CRL
11,372) were provided from the American
Type Culture Collection. Those used in the
experiment were derived from passages 5
and 6. The cell monolayers were cultured
in Modified Eagle’s medium (MEM, Gibco,
Invitrogen Corporation, Carlsbad, CA,
USA) and Ham’s F12 medium (HAM-
F12) (Cultilab, Campinas, SP, Brazil) (1 : 1)
supplemented with 10% of fetal bovine
serum (FBS, Gibco BRL, Campinas, Brazil),
and 1% of antibiotics–antimycotic (penicillin
G sodium – 100 U/ml), streptomycin –
100 mg/ml and amphotericin B – 0.025 mg/
ml; Gibco BRL). The cells were maintained
in a humidified incubator with 5% CO2 at
371C. When cells reached confluency they
were subcultivated using 0.1% of trypsin
(Sigma, St. Louis, MO, USA; 5 mg/ml) and
0.1% of calcium- and magnesium-free Dulbecco’s
phosphate-buffer saline solution
(Gibco, Invitrogen Corporation).
<strong>Platelet</strong> collection
Plasma was obtained from the venous
blood of a healthy male volunteer. Blood
was drawn into a 5 ml Vacutainer s (BD,
Curitiba, Paraná, Brazil) containing 500 ml
of the anticoagulant sodium citrate. PRP
was obtained following the protocol developed
by Macedo et al. (2003). Briefly, blood
was centrifuged twice at 120 g in an
ALC centrifuge (Centrifugette 4206 ALC,
ACE Surgical Supply Company Inc.,
Brockton, MA, USA) for 10 min at 201C
to remove red blood cells (first time) to
obtain the PRP (second time). Three hours
later, the PRP was diluted in the cell
culture medium for proliferative assessment
of the osteoblasts. A previous study
revealed that the activity of growth factors
present in PRP decreased 4 h after blood
was drawn (Ledent et al. 1995). Consequently,
the PRP used here was obtained
approximately 3 h after blood collection.
This procedure was repeated three times,
in a weekly interval, to obtain mean va-
Ferreira et al . Human osteoblasts growth
lues. Therefore, the blood was drawn three
times from the same male volunteer.
Evaluation of human osteoblast
proliferation by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl
tetrazolium bromide
(MTT) assay
hFOB1.19 cell cultures (3 10 4 cells/well)
were prepared in 96-well tissue culture
plates (Corning, NY, USA). After a 24 h
period of incubation, the cell culture medium
was replaced by one containing 50%,
25%, 12.5% and 6.125% of diluted PRP
without or with 10% FBS.
Next, platelet gel was obtained upon
addition of 5% calcium chloride to the
medium. Four days after replacing original
culture medium with another one containing
PRP, the MTT method (Mossman 1983)
was applied with modifications (Andrighetti-Fröhner
et al. 2003). After 4 days, at
371C inhumidified5%CO2atmosphere, 50 ml of MTT (Sigma, 1 mg/ml) solution
prepared in MEM : HAM-F12 (1 : 1) cell
medium was added to each well and the
plates were incubated for 4 h at 371C. Next,
the medium was not removed with suction,
and 100 ml of dimethyl sulfoxide (Merck
Biosciences, Darmstadt, Germany) was
added to each well to dissolve formazan
crystals. After gently shaking the plates for
5 min, whereby crystals were completely
dissolved, the absorbance was read on a
multiwell spectrophotometer (Bio-Tek, Elx
800, Winooski, VT, USA) at 540 nm. The
original MTT technique (Mossman 1983)
was modified, the cell culture medium was
not removed, for a gel clot was formed inside
the wells, and, by means of light microscopy,
the cells had migrated to the clot.
Afterwards, the <strong>influence</strong> of the PRP was
assessed by a scanning spectrophotometer
(Ultrospec 3000-Pharmacia Bio-tek, Winooski,
VT, USA) in the four concentrations
used in this research. This evaluation
showed no <strong>influence</strong> of the PRP in the cell
culture medium when read at 540 nm.
Therefore, PRP did not <strong>influence</strong> the results
obtained from the spectrophotometer (Bio-
Tek, Elx 800) at 540 nm. The percentage of
cell growth was calculated as [(A B)/
A 100], where A and B are the absorbances
of control and treated cells, respectively.
In this calculation, B was subtracted
from A and divided by the absorbance of the
treated group multiplied by 100, resulting in
the percentage of cell growth.
457 | Clin. Oral Impl. Res. 16, 2005 / 456–460

Ferreira et al . Human osteoblasts growth
The percentages of cell growth by the
PRP in relation to hFOB1.19 osteoblasts
represent mean standard error of the
mean values of three different experiments.
Variance analysis and the Tukey test
(Po0.05) were carried out as appropriate.
Results
Evaluation of human osteoblast
proliferation by MTT assay
The results of the <strong>influence</strong> of different
concentrations of PRP on hFOB1.19 osteoblast
proliferation in MEM : HAM-F12
(1 : 1) without and with 10% FBS are expressed
as percentages of cell growth that
indicate mean values of three independent
experiments. The results were evaluated
statistically using analysis of variance and
Tukey analysis (Fig. 1).
A comparison of the results of hFOB1.19
osteoblast proliferation by PRP with and
without 10% FBS showed that cell growth
increases as a function of the concentration
of PRP. Cell growth is always higher in the
groups receiving 10% FBS, showing the
highest <strong>influence</strong> on cell growth between
675.7% and 824% with 695.7% as the
median value for the groups receiving 50%
PRP. Figure 1 shows the results of the
variability considered in this research for
the groups receiving 10% FBS and those
not receiving FBS.
Discussion
Past investigations have established the
<strong>influence</strong> of growth factors on cell prolif-
Mean cell growth
900
800
700
600
500
400
300
200
100
0
10%
0%
256.9 ± 26.9
148.3 ± 20.7
191.2 ± 20.6
116.1 ± 10.4
eration (Gronthos & Simmons 1995; Kuznetov
et al. 1997; Lind 1998; Tian et al.
1999). PRP, which contains many growth
factors, has been shown to stimulate cell
proliferation in vitro (Slater et al. 1995;
Nakanishi et al. 1997; Weib<strong>rich</strong> et al.
2002; Kawasa et al. 2003; Lucarelli et al.
2003; Okuda et al. 2003). Whitman et al.
(1997) described the preparation of platelet
gel showing its advantages in bone graft
procedures to accelerate the healing process.
Literature on bone cell physiology
shows many instances where PRP was in
association with bone graft procedures. In
these cases, PRP has demonstrated an acceleration
of the formation of mature bone.
In addition, a greater formation of cancellous
bone was noted when compared
with bone grafts that did not receive
PRP when evaluated 4–6 months after the
procedure (Marx & Garg 2003). In vitro
investigations have identified that the
PDGF, a subcomponent of the PRP, has
a significant effect on cell proliferation
(Nakanishi et al. 1997; Weib<strong>rich</strong> et al.
2002; Kawase et al. 2003; Okuda et al.
2003).
However, questions are raised about the
efficacy of PRP in dental applications.
There are clinical studies where PRP was
used for grafting the floor of the maxillary
sinus in association with bone graft material
for increasing alveolar bone height prior
to the placement of endosseous dental
implants in the posterior maxilla. To
determine the efficacy of the sinus
augmentation procedure compared with
the results achieved with various surgical
techniques, grafting materials and im-
444.1 ± 23.1
433.5 ± 31.9
731.8 ± 46.5
666.4 ± 20.4
6.125 12.5 25 50
PRP concentration (%)
Fig. 1. The <strong>influence</strong> of different concentrations of platelet-<strong>rich</strong> <strong>plasma</strong> (PRP) on hFOB1.19 osteoblast
proliferation in MEM : HAM-F12 (1 : 1) medium without (0%) and with 10% of fetal bovine serum, expressed
as cell growth percentages. The mean values are presented on top of the bars with the standard error value.
458 | Clin. Oral Impl. Res. 16, 2005 / 456–460
plants, a systematic review of clinical studies
was conducted by Wallace & Froum
(2003). In this investigation, the authors
verified the effect on implant survival of
maxillary sinus augmentation vs. implant
placement in the non-grafted posterior
maxilla. The authors concluded that there
were insufficient data to recommend the
use of PRP in sinus graft surgery, a very
common procedure used in implant
dentistry.
In another investigation, the effect of
PRP on bone regeneration associated with
autogenous bone graft was evaluated in
animal models where bone defects received
autogenous bone in association with PRP,
and without PRP. Results of these studies
showed that the addition of PRP does not
appear to enhance new bone formation in
autogenous bone grafts (Schlegel et al.
2003, 2004; Choi et al. 2004; Wiltfang
et al. 2004).
Further, PRP was studied on bone augmentation
procedures in vitro and was
compared with bone morphogenetic protein
when added to autologous bone grafts.
The authors concluded that, by means of
a histomorphometric study, PRP failed
to enhance bone formation (Roldan et al.
2004).
Froum et al. (2002) studied the efficacy
of PRP on bone growth and osseointegration
in human maxillary sinus grafts when
associated with grafts of anorganic bovine
bone that contained minimal or no autogenous
bone. The results of their studies
showed that the effect of PRP did not show
a significant difference either in vital bone
production or in interfacial bone contact on
the test implants.
The studies enumerated above suggest
uncertainty in recommending the use of
PRP in bone-regeneration procedures.
However, the present investigation shows
in vitro that PRP has a positive effect on
osteoblast proliferation.
The results of this investigation are in
accordance with the results obtained by
Nakanishi et al. (1997) and Kawase et al.
(2003) showing that PRP stimulates cell
proliferation when added to culture medium
containing osteoblasts. In this study,
we observed a positive correlation between
the increased concentration of PRP in culture
medium and the rate of osteoblasts
proliferation, consistent with the results
published by Lucarelli et al. (2003), which

showed that 10% of PRP added to osteoblast
culture medium was sufficient to
induce evident cell proliferation. The present
study showed that 50% PRP caused
the best proliferative results in osteoblast
culture. Further studies are needed to establish
if progressively higher concentrations
of PRP would have a corresponding
<strong>influence</strong> on proliferation. In addition, our
results showed that FBS is not necessary for
PRP-mediated induction of hFOB1.19 osteoblast
proliferation as shown by Lucarelli
et al. (2003).
The content of PDGF and TGF-b present
in platelet gel was studied during the preparation
and storage of PRP. The content of
PDGF and TGF-b decreases according to
higher storage periods, showing less cell
growth promotion 4 h to 3 days after blood
is drawn. In this study, the experimental
use of blood to supplement the cell growth
media did not exceed 3 h after blood was
drawn (Ledent et al. 1995).
Currently, cultivation of human osteoblasts
in vitro is challenging (Lind 1998),
and PRP has demonstrated a significant
<strong>influence</strong> on osteoblast reproduction.
The positive results of in vitro
(Nakanishi et al. 1997; Weib<strong>rich</strong> et al.
2002; Lucarelli et al. 2003) studies encourage
research into further refinement
of methods for osteoblast cultivation.
Methods conducive to high yields of
osteoblast proliferation may contribute
ultimately to the development of processes
that promote growth or replacement
of substitute bone material. They also
could potentially have far-reaching benefits
in orthopedics, maxillofacial surgery as
well as implant dentistry by eliminating
the need for surgically complex bone harvesting
as well as reducing associated
pain and costs. However, the effects of
PRP associated with autogenous bone
grafts in clinical situations require further
investigation.
The purpose of the present study was
to evaluate the <strong>influence</strong> of progressively
higher concentrations of PRP on cell
growth medium. The maximum amount
of PRP added to the growth medium was
50%, showing the best proliferative results
in human osteoblast culture. Therefore,
further studies are suggested to answer
if higher concentrations of PRP would
alter the proliferative rate of human
osteoblasts.
Acknowledgements: The authors are
grateful to Prof. Dr Zenilda Laurita
Bouzon from the Department of Cell
Biology, Embryology and Genetics of
the University of Santa Catarina for her
assistance during the experimental part
of the research; to Esther Takamori,
MSc (Faculty of Dentistry, Bauru, SP)
for her assistance during laboratory
training and to Kenneth Faulkner
Weaver for his editorial assistance.
Résumé
Le but de cette étudeaété d’évaluer l’<strong>influence</strong> de
concentrations progressivement plus importantes de
<strong>plasma</strong> <strong>rich</strong>e en plaquettes (PRP) sur la prolifération
des ostéoblastes hFOB1.19 humain. Le PRP a été
obtenu de source humaine. Deux expériences ont été
conduites. Dans la première, le PRP a été dilué à 50,
25, 12,5 et 6,125 (v/v) avec un milieu de culture
(MEM : HAM-F12 et 1% d’antibiotiques-antimicotique)
avec 10% de sérum bovin foetal (FBS). Dans la
seconde expérience, toutes ces conditions étaient
identiques sauf qu’il n’y avait pas de FBS dans le
milieu de culture. Les résultats du test de la prolifération
des ostéoblastes étaient plus élevés quand ils
étaient stimulés par une dilution PRP de 50% avec
ou sans FBS. Une étude supplémentaireaété suggérée
pour déterminer si les concentrations supérieures
à 50% pouvaient apporter des taux plus élevés
de prolifération des ostéoblastes. Dans l’étude présente,
les résultats n’étaient pas différents (Po0,05)
avec des dilutions de PRP à 12,5 et 6,125%. De plus,
il était constaté que le FBS n’était pas nécessaire
pour l’induction de la prolifération des ostéoblastes
par PRP. Le PRP améliore donc la prolifération des
ostéoblastes et propose son application clinique dans
les processus de greffe osseuse en chirurgie buccale
implantaire.
Zusammenfassung
Der Einfluss von plättchenreichem Plasma auf das
Wachstum von menschlichen Osteoblasten
Ziel: Es sollte der Einfluss von progressiv erhöhten
Konzentrationen von plättchenreichem Plasma
(PRP) auf die Proliferation von menschlichen Osteoblsaten
hfOB1.19 untersucht werden.
Material und Methoden: Das PRP wurde bei
Menschen gewonnen. Zwei Experimente wurden
durchgeführt. Beim ersten wurde das PRP mit Kulturmedium
(MEM:HAM-F12 und 1% Antibiotika-
Antimykotika) auf 50, 25, 12.5 und 6.125 (v/v)
verdünnt und mit 10% fetalem bovinem Serum
(FBS) ergänzt. Beim zweiten Experiment waren alle
Bedingungen identisch, nur das FBS fehlte im Kulturmedium.
Resultate: Die Proliferation der Osteoblasten war
höher, wenn sie durch eine 50% PRP Verdünnung
mit oder ohne FBS stimuliert wurde. Es wird eine
weitere Studie vorgeschlagen, um zu bestimmen, ob
Konzentrationen über 50% höhere Proliferationsraten
der Osteoblasten zu Folge haben. In dieser
Untersuchung waren die Resultate mit 12.5 und
6.125% Verdünnungen nicht statistisch signifikant
verschieden (Po0.05). Zusätzlich konnte gezeigt
werden, dass FBS für die PRP gesteuerte Induktion
der Osteoblastenproliferation nicht nötig ist.
Schlussfolgerung: Diese Studie führt zur Schlussfolgerung,
dass PRP die Osteoblastenproliferation
fördert. Es wird die klinische Anwendung von PRP
bei Knochentransplantationen in Zusammenhang
mit dentalen Implantaten vorgeschlagen.
Resumen
Ferreira et al . Human osteoblasts growth
Objetivo: Evaluar la influencia de altas concentraciones
progresivamente de <strong>plasma</strong> rico en plaquetas
(PRP) en la proliferación de osteoblastos humanos
hFOB1.19.
Material y métodos: El PRP se obtuvo de fuentes
humanas. Se llevaron a cabo dos experimentos. En el
primero, el PRP se diluyó a 50, 25, 12.5 y 6.125 (v/v)
con medio de cultivo (MEM : HAM-F12 y 1% antibióticos-antimicóticos)
suplementado con 10% de
suero fetal bovino (FBS). En el segundo experimento,
todas las condiciones fueron idénticas salvo por la
ausencia de FBS en el medio de cultivo.
Resultados: Losresultadosdelapruebadeproliferación
de los osteoblastos fueron mayores cuando se
estimularon con la dilución de PRP al 50% con o sin
FBS. Se sugiere un estudio ulterior para determinar si
concentraciones por encima del 50% pueden causar
mayores índices de proliferación de osteoblastos. En
este estudio, los resultados no fueron estadísticamente
diferentes (Po0.005) con diluciones de PRP
de 12.5 y 6.125. Adicionalmente, se mostró que el
FBS no es necesario para la inducción mediada por
PRPdelaproliferación de osteoblastos.
Conclusión: Este estudio concluye que el PRP promueve
la proliferación de osteoblastos y sugirió la su
aplicación clínica para procedimientos de injerto
óseo en odontología deimplantes.
459 | Clin. Oral Impl. Res. 16, 2005 / 456–460

Ferreira et al . Human osteoblasts growth
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