See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/10639447

Platelet-Rich Plasma Contains High Levels of Platelet-Derived Growth Factor and

Transforming Growth Factor-?? and Modulates the Proliferation of Periodontally Related
Cells In Vitr...

Article  in  Journal of Periodontology · July 2003

DOI: 10.1902/jop.2003.74.6.849 · Source: PubMed

CITATIONS READS

275 1,134

8 authors, including:

Tomoyuki Kawase Hiromasa Yoshie

Niigata University Niigata University
127 PUBLICATIONS   2,606 CITATIONS    223 PUBLICATIONS   6,881 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

platelet concentrates View project

Periodontal Regeneration View project

All content following this page was uploaded by Tomoyuki Kawase on 20 May 2014.

The user has requested enhancement of the downloaded file.

J Periodontol • June 2003

Platelet-Rich Plasma Contains High Levels

of Platelet-Derived Growth Factor and
Transforming Growth Factor-␤ and
Modulates the Proliferation of Periodontally
Related Cells In Vitro
Kazuhiro Okuda,* Tomoyuki Kawase,† Manabu Momose,* Masashi Murata,* Yoshinori Saito,*
Hironobu Suzuki,* Larry F. Wolff,‡ and Hiromasa Yoshie*

Background: Platelet-rich plasma (PRP) is a fraction of plasma, in which

platelet-derived growth factor (PDGF) and transforming growth factor-β
(TGF-β) are thought to be concentrated. It is plausible that topically-applied
PRP up-regulates cellular activity and subsequently promotes periodontal
regeneration in vivo. However, the concentrations of these growth factors
in PRP have not been specifically determined and the biological effects of
PRP at the cellular and molecular levels have not been determined.

S
everal polypeptide
Methods: PRP obtained from 20 healthy subjects was prepared from
growth factors (e.g.,
plasma by centrifugation. These PRP preparations were immediately subjected
fibroblast growth fac-
to an evaluation for PDGF-AB and TGF-β1 using enzyme-linked immunosor-
tor, insulin-like growth fac-
bent assay (ELISA) kits. The biological effects of the PRP preparations were
tor-I, platelet-derived growth
evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament
factor [PDGF], transforming
cells. Cellular mitogenic activity was evaluated by counting cell numbers
growth factor-β [TGF-β], and
or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of
alkaline phosphatase (ALP) was immunocytochemically evaluated. bone morphogenetic proteins)
Results: In the PRP preparations, platelets were concentrated up to 70.9 × alone or in combination have
104 cells/µl (283.4% of the unconcentrated plasma). The levels of PDGF- been demonstrated to be
AB and TGF-β1 were also concentrated up to 182.0 ng/ml (440.6%) and effective on cell proliferation,
140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant cor- chemotaxis, differentiation,
relations between platelet counts and levels of these growth factors. PRP and extracellular matrix syn-
stimulated osteoblastic DNA synthesis and cell division (138% of control), thesis and consequently facil-
with simultaneous down-regulation of ALP, but suppressed epithelial cell itate periodontal repair and/
division (80% of control). PRP also stimulated DNA synthesis in gingival or regeneration in animal and
fibroblasts and periodontal ligament cells. human studies.1-10 Among
Conclusions: These data demonstrated that both PDGF-AB and TGF-β1 those growth factors, both
were highly concentrated in the PRP preparations. It is suggested PRP mod- PDGF and TGF-β have been
ulates cell proliferation in a cell type-specific manner similar to what has studied most extensively.
been observed with TGF-β1. Since synchronized behavior of related cell PDGF is stored in the bone
types is thought to be required for successful periodontal regeneration, it matrix11,12 and released upon
is further suggested these cell type-specific actions may be beneficial for activation of osteoblasts, re-
periodontal regenerative therapy. J Periodontol 2003;74:849-857. sulting in an increase of new
bone formation.13,14 In sup-
KEY WORDS
port of these findings, in vitro
Growth factors, platelet-derived; growth factors, transforming; studies demonstrated that
osteoblasts; periodontal ligament/anatomy and histology; plasma, PDGF initially stimulates bone
platelet-rich. resorption and also stimulates
the proliferation and chemo-
* Division of Periodontology, Department of Oral Biological Science, Course for Oral Life Science, Niigata
University Graduate School of Medical and Dental Sciences, Niigata, Japan.
taxis of osteoblasts.15,16 Thus,
† Division of Cellular Pharmacology, Department of Signal Transduction Research, Course for Molecular and it is generally accepted that
Cellular Medicine, Niigata University Graduate School of Medical and Dental Sciences.
‡ Division of Periodontology, Department of Preventive Sciences, University of Minnesota School of Dentistry,
PDGF functions as an ana-
Minneapolis, MN. bolic factor in bone metabo-

849
Growth Factors in PRP and Its Growth Modulatory Actions
lism.1,2,10 On the other hand, TGF-β is also found at
higher levels in bone matrix and upon activation facil-
itates wound healing under inflammatory conditions.17
In previous in vivo studies from our research group,3
we have shown that TGF-β1, when coated onto β-tri-
calcium phosphate pellets, substantially stimulates cell
proliferation and differentiation of osteoblast-lineage
cells, and eventually induces new bone formation in
experimental rat calvariae osseous defects. In addi-
tion, in association with gingival wound healing after
flap surgery in rats, we demonstrated that topically
applied TGF-β1 stimulates the proliferation of gingival
fibroblastic cells, the formation of blood vessels, and
the remodeling of extracellular matrix molecules,
effects which result in increased formation of granu-
lation tissue of the periodontal tissue.18
For economical and biological reasons, PRP prepa-
ration has been fractionated from plasma of patients
and used as an autologous source of these growth fac-
tors to regenerate periodontal tissue defects in the same
patients. This is based on evidence that human platelets
are enriched with PDGF and TGF-β.19 However, limited
information is currently available concerning growth
factor contents in PRP and their biological effects at the
cellular and molecular levels. Therefore, to provide clear
evidence for the clinical use of PRP, we have evaluated
the levels of PDGF and TGF-β in our PRP preparations
and examined the biological effects of PRP on the pro-
liferation of gingival fibroblastic, oral epithelial, osteoblas-
tic and periodontal ligament (PDL) cells in vitro.
MATERIALS AND METHODS
Collection and Preparation of PRP
Twenty healthy and non-smoking volunteers aged 24 to
48 years (8 females and 12 males, mean age = 35.1 ±
8.3 years) participated in this study. Prior to the study,
the volunteers were informed of the purpose and were
required to give informed consent. Seventeen ml of
whole blood were drawn from each subject and equally
aliquoted into two 10 ml sterile tubes coated with an
anti-coagulant (acid-citrate-dextrose). One tube (8.5 ml)
was used for PRP preparation while the whole blood in
the other tube was evaluated to determine the platelet
count and growth factors within the unconcentrated
plasma sample. PRP was prepared according to the
following procedure immediately after being drawn from
the subject and as described in the manufacturer’s
instructions.§ At the first centrifugation step (2,400
rpms for 10 minutes), PRP and platelet-poor plasma
(PPP) portions were separated from the red blood cell
(RBC) fraction. After discarding the RBC fraction, the
samples were again centrifuged (3,600 rpms for 15
minutes) to separate the PRP (0.6 ml) from PPP.
Determination of PDGF and TGF-β Levels
The resulting PRP preparations along with the uncon-
centrated plasma samples were immediately subjected
850
Volume 74 • Number 6
to the enzyme-linked immunosorbent assay (ELISA)

for evaluation of human PDGF-AB and TGF-β1. The
PDGF-AB levels were determined by a sandwich
ELISA method. According to the manufacturer’s pro-
tocol, 200 µl aliquots of each sample in duplicate were
applied to the microtiter plate pre-coated with a mon-
oclonal antibody against PDGF-AA, incubated for
2 hours at 22°C, probed with horseradish peroxidase
(HRP)-conjugated polyclonal antibody against PDGF-
BB for 2 hours at 22°C, and finally reacted with sub-
strate for 20 minutes at 22°C. At the end of the last
incubation, the reaction was stopped and the plates
were measured with a microplate reader¶ at 450 nm.
PDGF-AB levels (ng/ml) in the samples were deter-
mined by a standard curve (8.4 to 1000 pg/ml) pre-
pared in parallel with the evaluation of authentic
PDGF-AB.
TGF-β1 levels were similarly determined with ELISA.
Briefly, the samples were applied to the plate pre-
coated with TGF-β receptor II, incubated for 3 hours
at 22°C, probed with HRP-conjugated polyclonal anti-
body against TGF-β1 at 22°C for 3 hours, and finally
reacted with substrate for 20 minutes at 22°C. The
reaction was then stopped and color development was
read in a microplate reader at 450 nm. The TGF-β1
levels (ng/mg) in the samples were determined by a
standard curve (7.0 to 1000 pg/ml) prepared in par-
allel with the evaluation of authentic TGF-β.
Cells and Cell Culture
Human gingival fibroblast Gin-1, osteoblast MG63,
squamous epithelial SCC25, and rat osteoblast
UMR106 cells were obtained from a commercial
source.# As previously described,20,21 these cells were
maintained in Dulbecco’s modified Eagle medium
(DMEM)** or DMEM/F12 (1:1)** supplemented with
10% fetal calf serum (FCS)†† in humidified 5% CO2
and 95% air at 37°C. The method of periodontal liga-
ment (PDL) cell harvesting and preparation from PDL
tissue has been described in detail earlier.20 Briefly,
cultures of PDL cells were harvested from an extracted
tooth that was removed for orthodontic reasons in a
14-year-old healthy volunteer. This procedure also
required periodontal surgical intervention. The PDL tis-
sue attached to this extracted tooth was removed by
a surgical scalpel, minced, placed in 35 mm culture
dishes‡‡ in DMEM supplemented with 10% FCS, 100
µg/ml penicillin G, 50 µg/ml gentamicin sulfate, and
0.3 µg/ml amphotericin B** and overlaid with sterile
cover slips. After incubation for 10 days at 37°C, the
§ Heraeus Labofuge 300, Kendro Laboratory Products, Osterrode, Germany.

R&B Systems, Minneapolis, MN.
¶ Labsystem Japan, Multiskan BICHROMATIC, Tokyo, Japan.
# Dainippon Pharmaceutic Co., Osaka, Japan.
** Life Technologies, Grand Island, NY.
†† Hyclone, Logan, UT.
‡‡ Falcon, Franklin Lakes, NJ.
J Periodontol • June 2003
minced PDL tissue residue was removed from the cul-
ture dish, and any cellular outgrowths were harvested
with a standard trypsin/EDTA procedure.20 These cells
were then passaged to plastic dishes and cultured in
fresh medium at 37°C. Cell passages 4 to 8 were used
for the experiments described here.
Assessment of Cell Proliferation and DNA
Synthesis
The PRP preparations were stored at −20°C until used.
Both UMR106 and SCC25 cells were seeded at a den-
sity of 2 × 105 cells/35-mm culture dish and then incu-
bated with 5% (v/v) PRP in medium containing 1%
FCS for 24 hours at 37°C. Cells were enzymatically
detached at the end of the incubation period, and cell
numbers were counted with a hemocytometer.
The other cells were seeded at densities of 1 × 105
(Gin-1) or 2 × 105 cells/35-mm dish (MG63 and PDL)
and treated with 2% PRP as described above. In the
presence of added PRP (≥0.5%), the cells became
incorporated into a gel-like material. Under these con-
ditions cells could not be detached without significant
damage or loss for cell counting, or were not suitable
for spectrophotometrical assay. Therefore to avoid
these disadvantages, we developed a new image-ana-
lytical method. Cells were labeled with 5-bromo-
deoxyuridine (BrdU) for an additional 15 minutes, har-
vested with a rubber policeman, and smeared onto
glass slides. Each specimen was fixed once with 3.7%
formaldehyde in a phosphate buffered solution (pH 7.4)
and subjected to immunocytochemical staining with
monoclonal anti-BrdU antibody and Cy3-conjugated
anti-mouse immunoglobulin G (IgG) antibody.§§ For
detection of cells, the specimens were also stained
with polyclonal anti-p38-mitogen-activated protein
kinase (MAPK) antibody

and conjugated anti-rabbit
IgG antibody.¶¶
Immunocytochemical Detection of Alkaline
Phosphatase
MG63 and UMR106 cells were also subjected to
immunocytochemical detection of alkaline phosphatase
(ALP) expression. As described above, cells were har-
vested, smeared, and stained with monoclonal anti-
ALP antibody## and Cy3-conjugated anti-mouse IgG.§§
Cell distribution was visualized with anti-p38-MAPK
antibody.
Statistical Analysis
Wilcoxon’s signed-rank matched pairs tests were used
for intra-group data set comparison between PRP and
the unconcentrated plasma sample within each vol-
unteer. Scatter plots and Pearson correlation coeffi-
cient (r) were used to demonstrate the relationship
between platelet counts in the PRP preparations (or
the unconcentrated plasma samples) and their respec-
tive growth factor levels. The null hypothesis was
Okuda, Kawase, Momose, et al.
rejected when the risk percentage was below 5% (P
<0.05). In the biological examination, statistical analy-
ses were performed using the Student t-test or ana-
lyzed by 1-way analysis of variance. Comparisons bet-
ween individual groups were made using Tukey’s
multiple comparison test; P values <0.05 were considered
significant.
RESULTS
Platelet Counts and Levels of Growth Factors
The platelet counts and growth factor levels in our PRP
preparations are shown in Figure 1. The platelet counts
in the PRP preparations ranged from 41.4 to 124.0 ×
104 platelets/µl, and the median and mean were 65.3 ×
104 platelets/µl and 70.9 ± 21.6 × 104 platelets/µl,
respectively. In contrast, the platelet counts in the
unconcentrated plasma ranged from 17.5 to 34.8 ×
104 platelets/µl, and the median and mean were 23.9 ×
104 platelets/µl and 25.7 ± 4.6 × 104 platelets/µl,
respectively. Thus, the percentage increase was cal-
culated as 283.4 ± 98.3% (the mean), with a range
from 148.2% to 525.4% (versus the unconcentrated
plasma; P <0.001) (Fig. 1A).
The PDGF-AB levels in the PRP preparations ranged
from 57.8 to 403.8 ng/ml, and the median and mean
were 166.1 ng/ml and 182.0 ± 75.5 ng/ml, respec-
tively. In contrast, in the unconcentrated plasma,
PDGF-AB levels ranged from 14.8 to 128.9 ng/ml, and
the median and mean were 38.6 ng/ml and 51.8 ± 33.4
ng/ml, respectively. Thus, the percentage increase was
determined as 440.6 ± 211.8% (the mean), with a
range from 191.0% to 914.7% (versus the unconcen-
trated plasma; P <0.001) (Fig. 1B).
The TGF-β1 level in the PRP preparations ranged
from 50.4 to 262.3 ng/ml, and the median and mean
were 126.2 and 140.9 ± 53.5 ng/ml, respectively. In
the unconcentrated plasma samples, in contrast, the
TGF-β1 levels ranged from 21.3 to 60.1 ng/ml, and
the median and mean were 42.4 and 41.6 ± 11.4 ng/
ml, respectively. Thus, the percentage increase
was determined as 346.6 ± 111.3% (the mean), with
a range from 158.7% to 538.9% (versus the uncon-
centrated plasma; P <0.001) (Fig. 1C). These results
indicate that those growth factors are significantly
concentrated along with platelets in the PRP prepa-
rations.
The correlation was then analyzed between platelet
counts and growth factor levels using scatter plots.
The Pearson correlation coefficient (r) values were
calculated as 0.48 (PDGF-AB: P <0.05) and 0.57
(TGF-β1: P <0.01). Therefore, significant correlations
between these 2 parameters were clearly demon-
§§ Amersham Pharmacia Biotech, Buckinghamshire, U.K.


New England Biolab, Beverly, MA.
¶¶ Molecular Probes, Eugene, OR.
## Chemicon International, Inc., Temecula, CA.
851
Growth Factors in PRP and Its Growth Modulatory Actions
Figure 1.
Changes in platelet counts and growth factor levels in the process of
PRP preparation. A. Platelet counts in PRP and unconcentrated plasma.
B. PDGF-AB level in PRP and unconcentrated plasma. C. TGF-β1 level
in PRP and unconcentrated plasma.
strated in the PRP preparations (Figs 2A and 2B). In
contrast, no significant correlations were found in the
unconcentrated plasma samples (Figs. 2C and 2D).
These data suggest that both PDGF-AB and TGF-β1
were mainly derived from platelets in the PRP prepa-
rations.
852
Volume 74 • Number 6
Effects of PRP on Cell Proliferation and Alkaline
Phosphatase (ALP) Expression
The effects of PRP on the proliferation of osteoblast
UMR106 and epithelial SCC25 cells are shown in Fig-
ure 3. Treatment with 5% PRP for 24 hours increased
the number of osteoblastic UMR106 cells by 138% (ver-
sus the cultures with no addition) (Fig. 3A), whereas
it decreased the number of epithelial SCC25 cells by
82% (versus the cultures with no addition) (Fig. 3B).
These data are essentially consistent with the obser-
vations from the DNA synthesis assay as described
below.
The effects of PRP on BrdU incorporation into
osteoblastic and epithelial cells are shown in Figure 4.
Treatment with 2% PRP for 24 hours (plus 15 minutes
for labeling) substantially increased the number of cells
positive for BrdU in UMR106, but decreased that in
SCC25 cells. This method was further applied to the
rest of the cell types. PRP treatment (2%, 24 hours)
again reproducibly increased BrdU incorporation into
MG63, Gin-1 and PDL cells. These data are summa-
rized in Table 1.
In the parallel experiment, the effects of 2% PRP on
ALP expression in MG63 cells were immunocyto-
chemically detected, as shown in Figure 5. PRP down-
regulated ALP protein expression in MG63 cells (Panel
D versus B). In data not shown, however, 2% PRP did
not influence ALP expression in the other osteoblastic
UMR106 cells.
DISCUSSION
PRP has been thought, but not well demonstrated, to
contain certain growth factors, such as PDGF and TGF-
β, at high concentrations. In addition, it has not been
demonstrated that these growth factors in PRP are
involved in accelerating regeneration of periodontal tis-
sue damaged by periodontitis. In the present study, we
have clearly demonstrated that PRP is a mixture of
concentrated platelets and growth factors, especially
PDGF and TGF-β. In addition, we have for the first time
shown that PRP containing these growth factors effi-
ciently and effectively regulated the proliferation of
periodontal related cells in culture.
Level of Platelets in PRP
To prepare PRP, in the current study, freshly collected
plasma (i.e., unconcentrated plasma) was centrifuged
at 2,400 rpms for 10 minutes as the first step and then,
after discarding the RBC fraction, at 3,600 rpms for
15 minutes as the second step. The platelet density in
the resulting PRP preparations was increased by 283.4%
when compared to the unconcentrated plasma. This
percentage increase in platelet density was higher
(205.7%) than that obtained by Landesberg and
coworkers,22 but lower (338%) than that obtained by
Marx and coworkers.23 This discrepancy could be due
to a variation of the procedures for centrifugation of the
J Periodontol • June 2003 Okuda, Kawase, Momose, et al.

Figure 2.
Relationship between platelet counts and growth factor levels in the PRP preparations. A. Scatter plot of PDGF-AB levels versus platelet counts in PRP.
B. Scatter plot of TGF-β1 levels versus platelet counts in PRP. C. Scatter plot of PDGF-AB levels versus platelet counts in unconcentrated plasma. D. Scatter
plot of TGF-β1 levels versus platelet counts in unconcentrated plasma.

original plasma samples (e.g., force, time, etc.). From
a qualitative point of view, however, our PRP prepara-
tions were nearly identical to those obtained by other
investigators and were applicable for the experiments
performed in this investigation.
Levels of PDGF and TGF-β in PRP
As described above, it has been postulated that the
levels of PDGF-AB and TGF-β1 in PRP preparations
depend on the number of platelets involved. In the pre-
sent study, we did demonstrate a statistically signifi-
cant correlation between the platelet counts and growth
factor (PDGF-AB and TGF-β1) levels in the PRP prepa-
rations. However, no statistically significant correlation
was found between these 2 parameters in the uncon-
centrated plasma. This result implies that the platelet
is the major source of these growth factors in the PRP.
Weibrich and colleagues24 have recently quantitated
concentrated levels of PDGF-AB and TGF-β1 in PRP
by 2 different methods. The platelet concentrate col-
lection system (PCCS) PRP method produced about
252 ng/ml of PDGF-AB, 467 ng/ml of TGF-β1, and
2.23 × 106 platelets/µl, while the curasan-type PRP kit
produced 314 ng/ml of PDGF-AB, 80 ng/ ml of TGF-

853
Growth Factors in PRP and Its Growth Modulatory Actions
Figure 3.
Effects of PRP on the proliferation of osteoblast and epithelial cells.
Both UMR106 (A) and SCC25 cells (B) were seeded at a density of
2 × 105 cells/35 mm culture dish and treated with 5% PRP for
24 hours in media supplemented with 1% FCS. At the end of the
incubation period, cells were harvested and counted with a
hemocytometer. P <0.01, P <0.002 versus each control.
β1, and 1.14 × 106 platelets/µl. Thus, PDGF-AB and
TGF-β1 levels are determined as 113.0 and 209.4
ng/109 platelets, respectively, in the PCCS method,
while those levels in the curasan-type system are 275.4
and 70.2 ng/109 platelets, respectively. In this study,
on the other hand, we found that PDGF-AB and TGF-
β1 levels in the PRP preparations were 256.7 and 198.7
854
Volume 74 • Number 6
Figure 4.
Effects of PRP on BrdU incorporation in osteoblastic, and epithelial
cells. Cells were seeded and treated with 5% PRP (UMR106 and
SCC25 cells) for 24 hours (Panels C and D). Non-treated cultures
served as controls (Panels A and B). For an additional 15 minutes,
cells were labeled with BrdU and then harvested for preparation of
smeared specimens. After fixation, cells were immunocytochemically
stained for evaluation of BrdU incorporation (Panels B and D). For
distribution of cells in a view (Panels A and C), the same specimens
were also stained for p38-MAPK. Each experiment was repeated a
minimum of 5 times and all experiments gave similar results.
ng/109 platelets, respectively. Compared to the above
data,24 our preparation method appeared more effi-
cient in simultaneously concentrating both growth fac-
tors. If our method is further improved to obtain higher
densities of platelets in PRP, it is theoretically possible
to provide these growth factors at higher levels than that
achieved at the present. The method for obtaining a
higher density of platelets in PRP is currently under
investigation in our laboratory.
It has also been thought that PDGF and TGF-β are
released and present at higher levels in areas where
J Periodontol • June 2003
Table 1.
Effects of PRP on BrdU Incorporation in
Osteoblastic, Epithelial, Fibroblastic, and
PDL Cells
Cell Type Regulation of BrdU Incorporation
UMR106 Up
SCC25 Down
MG63 Up
Gin-1 Up
PDL Up
Figure 5.
Effects of ALP expression on MG63 cells. Cells were treated with 2% PRP
for 24 hours (Panels C and D) and smeared for immunocytochemical
staining of ALP (Panels B and D) or p38-MAPK (Panels A and C).
Non-treated cultures served as controls (Panels A and B).
wound healing is occurring.25 According to previous
reports, PDGF levels in circulating platelets are in the
order of 0.06 ng per 1 × 106 platelets,26 and the effec-
tive concentration of TGF-β, when exogenously applied
in vitro and in vivo, ranges from 10 ng/ml to 1 µg/
ml.3,27,28 Therefore, our data support the notion that
the growth factor levels in our PRP preparations are
above the minimum essential level to induce the bio-
logical effects necessary for periodontal wound heal-
ing and tissue repair/regeneration. To further investi-
gate this concept, we have examined the effects of
PRP on cultured cells.
Biological Action of PRP on Periodontally
Associated Cells In Vitro
Our data suggest that PRP acts as a mitogen on
osteoblastic (UMR106) cells, but acts as a growth
inhibitor on epithelial (SCC25) cells. These observa-
Okuda, Kawase, Momose, et al.
tions were reconfirmed by our image-analytical method
for DNA synthesis. When this method was further applied
to the other cell types, PRP appeared to act as a mito-
gen also on osteoblastic MG63, gingival fibroblastic
Gin-1 and PDL cells.
These biological actions seem very similar to pre-
viously published data. PDGF15,16 and TGF-β29 stim-
ulate mitogenic activity in osteoblasts, but TGF-β acts
as a growth inhibitor for epithelial cells.30-32 Also in
PDL29,33 and gingival fibroblastic cells,26,33 PDGF
and/or TGF-β1 have been shown to stimulate the pro-
liferation of these cell types. Taken together with our
data on growth factor levels, these findings suggest
that PRP possibly modulates cell proliferation by PDGF-
and/or TGF-β-mediated mechanisms.
We believe that PRP actions are “essentially” iden-
tical to TGF-β actions. Compared to authentic TGF-
β1 alone, however, our PRP preparations more potently
stimulated PDL and osteoblastic proliferation and inhib-
ited epithelial proliferation (data not shown). Thus, we
speculate that other identified (e.g., PDGF) and uniden-
tified components in PRP may effectively function in
combination with TGF-β in the periodontal regeneration
process. In a separate investigation,34 we have demon-
strated that fibrinogen contained in PRP is converted to
insoluble fibrin and potently stimulated collagen syn-
thesis in PDL and osteoblastic cells. Thus, it is also pos-
sible that fibrin converted from fibrinogen could work in
concert with these growth factors in promoting regen-
eration. At present time, we have no direct evidence to
support this kind of synergistic action. However, our
laboratory is continuing to investigate the potential
benefits of using PRP in periodontal regeneration.
In conclusion, these data demonstrated that PRP
can serve as a source for the representative growth
factors such as PDGF and TGF-β. In addition, PRP also
modulates cell proliferation in a cell type-specific man-
ner. Its ability to suppress epithelial cell proliferation
seems advantageous for periodontal regeneration. The
suppression of the down-growth of junctional epithe-
lium onto dental root surfaces in the regeneration
process would avoid intereference by epithelium with
the formation of a new connective tissue attachment
on the root surface. These biological actions are very
similar to previous reports on PDGF and TGF-
β.15,16,26,29-33 Our results, therefore, provide convinc-
ing evidence for the clinical application of PRP as a
potent tool to facilitate periodontal regeneration.
ACKNOWLEDGMENTS
This study was supported in part by Grants-in Aid for
Scientific Research 14571979 and 14771216 from the
Japan Society for the Promotion of Science.
REFERENCES
1. Lynch SE, Williams RC, Polson AM, et al. A combina-
tion of platelet-derived and insulin-like growth factors
855
Growth Factors in PRP and Its Growth Modulatory Actions
enhances periodontal regeneration. J Clin Periodontol
1989;16:545-548.
2. Wang H-L, Pappert TD, Castelli WA, Chiego DJ Jr, Shyr
Y, Smith BA. The effect of platelet-derived growth fac-
tor on the cellular response of the periodontium: An
autoradiographic study on dogs. J Periodontol 1994;65:
429-436.
3. Okuda K, Nakajima Y, Irie K, et al. Transforming growth
factor-β1 coated β-tricalcium phosphate pellets stimulate
healing of experimental bone defects of rat calvariae.
Oral Dis 1995;1:92-97.
4. Mohammed S, Pack ARC, Kardos TB. The effect of
transforming growth factor beta one (TGF-β1) on wound
healing, with or without barrier membranes, in a class
II furcation defect in sheep. J Periodont Res 1998;33:335-
344.
5. Takayama S, Murakami S, Shimabukuro Y, Kitamura
M, Okada H. Periodontal regeneration by FGF-2
(bFGF) in primate models. J Dent Res 2001;80:2075-
2079.
6. Ripamonti U, Heliotis M, van den Heever B, Reddi AH.
Bone morphogenetic proteins induce periodontal regen-
eration in the baboon (Papio ursinus). J Periodont Res
1994;29:439-445.
7. Bowers G, Felton F, Middleton C, et al. Histologic com-
parison of regeneration in human intrabony defects when
osteogenin is combined with demineralized freeze-dried
bone allograft and with purified bovine collagen. J Perio-
dontol 1991;62:690-702.
8. Giannobile WV, Ryan S, Shih M-S, Su DL, Kaplan PL,
Chan TCK. Recombinant human osteogenic protein-1
(OP-1) stimulates periodontal wound healing in class III
furcation defects. J Periodontol 1998;69:129-137.
9. Sigurdsson TJ, Lee MB, Kubota K, Turek TJ, Wozney JM,
Wikesjö UME. Periodontal repair in dogs: Recombinant
human bone morphogenetic protein-2 significantly
enhances periodontal regeneration. J Periodontol 1995;
66:131-138.
10. Howell TH, Fiorellini JP, Paquette DW, Offenbacher S,
Giannobile WV, Lynch SE. A phase I/II clinical trial to
evaluate a combination of recombinant human platelet-
derived growth factor-BB and recombinant human
insulin-like growth factor-I in patients with periodontal
disease. J Periodontol 1997;68:1186-1193.
11. Zhang L, Leeman E, Carnes DC, Graves DT. Human
osteoblasts synthesize and respond to platelet-derived
growth factor. Am J Physiol 1991;261:C348-C354.
12. Rydziel S, Ladd C, McCarthy TL, Centrella M, Canalis E.
Determination and expression of platelet-derived growth
factor-AA in bone cell cultures. Endocrinology 1992;130:
1916-1922.
13. Canalis E, McCarthy TL, Centrella M. Effects of platelet-
derived growth factor on bone formation in vitro. J Cell
Physiol 1989;140:530-537.
14. Cochran DL, Rouse CA, Lynch SE, Graves DT. Effects
of platelet-derived growth factor isoforms on calcium
release from neonatal mouse calvariae. Bone 1993;
14:53-58.
15. Gilardetti RS, Chaibi MS, Stroumza J, et al. High-affin-
ity binding of PDGF-AA and PDGF-BB to normal human
osteoblastic cells and modulation by interleukin-1. Am
J Physiol 1991;261:C980-C985.
16. Hughes FJ, Aubin JE, Heersche JNM. Differential
chemotactic responses of different populations of fetal
856
Volume 74 • Number 6
rat calvaria cells to platelet-derived growth factor and
transforming growth factor β. Bone Miner 1992;19: 63-
74.
17. Sporn MB, Roberts AB. Transforming growth factor-β:
Recent progress and new challenges. J Cell Biol 1992;
119:1017-1021.
18. Okuda K, Murata M, Sugimoto M, et al. TGF-β1 influ-
ences early gingival wound healing in rats: an immuno-
histochemical evaluation of stromal remodelling by extra-
cellular matrix molecules and PCNA. J Oral Pathol Med
1998;27:463-469.
19. Assoian RK, Grotendorst GR, Miller DM, Sporn MB. Cel-
lular transformation by coordinated action of three pep-
tide growth factors from human platelets. Nature 1984;
309:804-806.
20. Kawase T, Okuda K, Yoshie H, Burns DM. Cytostatic
action of enamel matrix derivative (EMDOGAIN) on
human oral squamous cell carcinoma-derived SCC25
epithelial cells. J Periodont Res 2000;35:291-300.
21. Kawase T, Okuda K, Momose M, Kato Y, Yoshie H, Burns
DM. Enamel matrix derivative (EMDOGAIN) rapidly
stimulates phosphorylation of the MAP kinase family
and nuclear accumulation of smad2 in both oral epithe-
lial and fibroblastic human cells. J Periodont Res 2001;
36:367-376.
22. Landesberg R, Roy M, Glickman RS. Quantification of
growth factor levels using simplified method of platelet-
rich plasma gel preparation. J Oral Maxillofac Surg 2000;
58:297-300.
23. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR,
Strauss JE, Georgeff KR. Platelet-rich plasma: Growth
factor enhancement for bone grafts. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 1998;85:638-646.
24. Weibrich G, Kleis WKG, Hafner G. Growth factor levels
in the platelet-rich plasma produced by 2 different meth-
ods: Curasan-type PRP kit versus PCCS PRP system. Int
J Oral Maxillofac Implants 2002;17:184-190.
25. Marx RE. Platelet-rich plasma: A source of multiple autol-
ogous growth factors for bone grafts. In: Lynch SE,
Genco RJ, Marx RE, eds. Tissue Engineering. Applica-
tions in Maxillofacial Surgery and Periodontics. Chicago:
Quintessence Publishing Co., Inc.; 1999:71-82.
26. Bowen-Pope DF, Vogel A, Ross R. Production of
platelet-derived growth factor-like molecules and
reduced expression of platelet-derived growth factor
receptors accompany transformation by a wide spec-
trum of agents. Proc Natl Acad Sci (USA) 1984;81:2396-
2400.
27. Alvares O, Klebe R, Grant G, Cochran DL. Growth fac-
tor effects on the expression of collagenase and TIMP-1
in periodontal ligament cells. J Periodontol 1995;66:
552-558.
28. Arnaud E, Morieux C, Wybier M, de Vernejoul MC. Poten-
tiation of transforming growth factor (TGF-β1) by nat-
ural coral and fibrin in a rabbit cranioplasty model. Cal-
cif Tissue Int 1994;54:493-498.
29. Piché JE, Graves DT. Study of the growth factor require-
ments of human bone-derived cells: A comparison with
human fibroblasts. Bone 1989;10:131-138.
30. Shipley GD, Pittelkow MR, Wille JJ Jr, Scott RE, Moses
HL. Reversible inhibition of normal human proker-
atinocyte proliferation by type β transforming growth
factor-growth inhibitor in serum free medium. Cancer
Res 1986;46:2068-2071.
 J Periodontol • June 2003 Okuda, Kawase, Momose, et al.

31. Tucker RF, Shipley GD, Moses HL, Holley RW. Growth Correspondence: Dr. Kazuhiro Okuda, Division of Periodon-
inhibitor from BSC-1 cells closely related to platelet type tology, Department of Oral Biological Science, Niigata Uni-
β transforming growth factor. Science 1984;226:705- versity Graduate School of Medical and Dental Sciences, 2-
707. 5274, Gakkocho-Dori, Niigata 951-8514, Japan. Fax: 81-
32. Kawase T, Okuda K, Yoshie H, Burns DM. Anti-TGF-β 25-227-0808; e-mail: okuda@dent.niigata-u.ac.jp.
antibody blocks enamel matrix derivative-induced upreg-
ulation of p21WAF1/cip1 and prevents its inhibition of Accepted for publication December 6, 2002.
human oral epithelial cell proliferation. J Periodont Res
2002;37:255-262.
33. Dennison DK, Vallone DR, Pinero GJ, Rittman B,
Caffesse RG. Differential effect of TGF-β1 and PDGF on
proliferation of periodontal ligament cells and gingival
fibroblasts. J Periodontol 1994;65:641-648.
34. Kawase T, Okuda K, Wolff LF, Yoshie H. Platelet-rich
plasma-derived fibrin clot formation stimulates collagen
synthesis in periodontal ligament and osteoblastic cells
in vitro. J Periodontol 2003;74:858-864.

857

View publication stats