966THE JOURNAL OF BONE AND JOINT SURGERY
The effect of the platelet concentration in
platelet-rich plasma gel on the regeneration
K. A. Siwicka,
University School of
? M. Kawasumi, MD,
? H. Kitoh, MD, PhD,
? K. A. Siwicka, MD,
? N. Ishiguro, MD, PhD,
Professor of Orthopaedics
Department of Orthopaedic
Nagoya University School of
Medicine, 65 Tsurumai-cho,
Showa-ku Nagoya, Aichi 466-
Correspondence should be sent
to Dr M. Kawasumi; e-mail:
©2008 British Editorial Society
of Bone and Joint Surgery
J Bone Joint Surg [Br]
Received 24 September 2007;
Accepted after revision
7 February 2008
The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation
and differentiation of rat bone-marrow cells and to determine an optimal platelet
concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded
in different concentrations of platelet-rich plasma gel were cultured for six days. Their
potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-
lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable
concentrations were transplanted into the distraction gap and the quality of the regenerate
bone was evaluated radiologically.
Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-
dependent manner. Although no significant differences in the production and mRNA
expression of alkaline phosphatase were detected among these groups, mature bone
regenerates were more prevalent in the group with the highest concentration of platelets.
Our results indicate that a high platelet concentration in the platelet-rich plasma in
combination with osteoblastic cells could accelerate the formation of new bone during
There is currently an increased interest in the
use of platelet-rich plasma for bone regenera-
tion and healing. It contains osteo-inductive
growth factors including platelet-derived
growth factors, vascular endothelial growth
factor, insulin-like growth factor and trans-
forming growth factors.1-5 These cause the
proliferation and differentiation of local
osteoprogenitor cells into bone-forming cells,
leading to mineralisation and the formation of
bone matrix.6,7 It has been shown, however,
that variations in the concentration of platelets
in the platelet-rich plasma have diverse effects
on the proliferation and differentiations of the
osteoblasts.8-14 Choi et al8 reported that the
viability and proliferation of alveolar bone
cells were suppressed by a high, and stimulated
by a low concentration of platelets in the
platelet-rich plasma. Uggeri et al14 found that
proteins released from platelet gel stimulated
the proliferation of osteoblasts in a dose-
dependent manner. In order to provide clear
evidence for the clinical use of platelet-rich
plasma, it is necessary to determine its direct
effect on osteogenic cells at the cellular and
Platelet-rich plasma is an autologous prepa-
ration and there are therefore no concerns
about the transmission of disease or an
immunogenic reaction. There have been
several clinical trials using a combination of
platelet-rich plasma and bone graft or
osteoprogenitor cells which have aimed at
increasing the rate of osteogenesis and the
enhancement of the formation of bone.4-19
We have established a new technique of
transplantation using culture-expanded bone-
marrow cells and platelet-rich plasma in distrac-
tion osteogenesis of the long bones and demon-
strated a satisfactory clinical outcome by
accelerating the formation of new bone.15-17 To
improve our combined bone-marrow cells and
platelet-rich plasma cell therapy further, it was
necessary to determine the optimal platelet con-
centration of the plasma for bone regeneration.
Our aim therefore was to evaluate the osteo-
genic differentiation of rat bone-marrow cells
embedded in platelet-rich plasma gels with dif-
ferent platelet concentrations. Using a rat
model of limb lengthening, the rat bone-
marrow cells and platelet-rich plasma gels were
then transplanted into the lengthened femur,
and bone formation in vivo was analysed.
Materials and Methods
Preparation of platelet-rich plasma gels with
different platelet concentrations. Under gen-
eral anaesthesia whole blood was withdrawn
THE EFFECT OF THE PLATELET CONCENTRATION IN PLATELET-RICH PLASMA GEL ON THE REGENERATION OF BONE 967
VOL. 90-B, No. 7, JULY 2008
by cardiac puncture from 90 14-week-old male Sprague-
Dawley rats (Japan SLC Inc., Shizuoka, Japan). For one
series, 40 ml of anticoagulated whole blood was initially
centrifuged at 1100 g for ten minutes to precipitate the red
blood-cell fraction. The supernatant was again centrifuged
to produce precipitation of platelets. The platelet-rich
plasma was then divided into three groups (low concen-
trate; medium; and high concentrate) according to the
amount of supernatant which had been removed. We used
platelet concentrations as suggested by Marx.1 The super-
natant alone was used as platelet-poor plasma. The number
of platelets in the high, medium, low concentrate, platelet-
poor plasma and whole blood was counted using a Sysmex
XE 2100 haematology analyser (Sysmex, Kobe, Japan).
Bone-marrow cells culture. Specially-prepared
essential medium alpha (Gibco-BRL, Carlsbad, California),
supplemented with 10% fetal bovine serum (Dainippon
Pharmaceutical, Osaka, Japan), 100 μl/ml of penicillin-
streptomycin (Gibco-BRL, Life Technologies, Grand Island,
New York), 0.2 mM ascorbic acid (Sigma, St Louis, Missouri)
10 mM Na-b-glycerophosphate (Sigma), and 10-8M
dexamethasone (Sigma), was used as a growth medium in all
the cell-culture experiments.
Rat bone-marrow cells were isolated from 60 four-week-
old, male Sprague-Dawley rats weighing between 90 g and
110 g using the technique described by Takamine et al.20
The harvested cells were cultured with 8 ml of growth
medium. The adherent cells were expanded as monolayer
cultures in a 5% CO2/95% air atmosphere at 37°C with
medium changes every three days. When the cultures
became nearly confluent, the cells were dissociated with
0.25% trypsin/ethylenediaminetetraacetic acid (EDTA)
(Gibco BRL) and re-seeded at a density of 3 × 105 cells/dish.
Cells passaged twice (P2) were used for all three-
dimensional (3-D) cultures.
3-D gel-embedded culture of rat bone-marrow cells. A sus-
pension of 8 × 104 rat bone-marrow cells in 150 μl of each
platelet derivative was mixed with 50 μl of a thrombin/
CaCl2 solution to obtain platelet gel. The rat bone-marrow
cells were also mixed with bovine-dermal-pepsin-solubi-
lised type-I collagen (Koken Co., Ltd, Tokyo, Japan) in gel
form at 37°C. The rat bone-marrow cells embedded in gel
at a density of 4 × 105 cells/ml were cultured for six days
and were then divided into five groups (group P, embedded
in the platelet-poor plasma gel; group L, embedded in the
low concentration gel; group M, embedded in the medium
concentration gel; group H, embedded in the high concen-
tration gel; and group C, embedded in the collagen gel).
In vitro cell proliferation. The WST-1 cell proliferation
reagent (Roche Diagnostics GmbH, Mannheim, Germany)
was used for counting the cells.11,21 The WST-1 test mea-
sures the mitochondrial activity which corresponds to the
number of viable cells. After incubation for one hour at
37°C, the absorption of the medium containing the WST-1
reagent was measured by an enzyme-linked immuno-
absorbent assay (ELISA) Reader (Thermo Fisher Scientific
Inc., Waltham, Massachsetts) at 440 nm with a reference
wavelength > 600 nm. The WST-1 assay was carried out at
intervals of two days after the initiation of the 3-D cultures.
Each experiment was repeated twice with 20 samples for
each group, resulting in a total of 40 samples per group.
In vitro osteogenic differentiation. The cultures were washed
in phosphate-buffered saline and the cells were lysed using
a UP50H ultrasonic processor (Hielscher Ultrasonics
GmbH, Teltow, Germany). Using the alkaline phospha-B-
test (Wako, Osaka, Japan), the activity of alkaline phos-
phatase in the cell lysates was measured every two days
after embedding of the cells.
After the gels had been dissolved by urokinase (Uronase;
Mochida, Tokyo, Japan) and collagenase (Sigma-Aldrich,
Tokyo, Japan), the total RNA was isolated every two days
using the RNeasy Mini Kit (QIAGEN KK, Tokyo, Japan).
For reverse transcription into circular DNA (cDNA)
(reverse transcription system; Perkin Elmer, Waltham, Mas-
sachusetts), one μg of RNA was used as a template. cDNA
was amplified by the polymerase chain reaction with oligo-
nucleotide primers for alkaline phosphatase and glyceralde-
hyde-3-phosphate dehydrogenase as housekeeping genes.22
Quantitative real-time was carried out using a Light Cycler
480 Real-Time System (Roche Diagnostics Corporation,
Indianapolis, Indiana) and a Light Cycler Fast Start DNA
Master SYBR Green I (Roche Diagnostics Corporation).
Rat model of limb lengthening. All the animal experiments
were carried out in compliance with the laws and guidelines
for the experimental use and care of animals. Application
of an external fixation device to the femora of 91 nine-
week-old male Sprague-Dawley rats weighing between
Day of culture
Relative proliferation (450 nm)
Graph showing the number of viable rat bone-marrow cells cultured
with platelet-rich plasma gels with different concentrations of platelets
and collagen gel. Cell proliferation was significantly enhanced in group
H. (P, platelet-poor plasma; L, low concentration platelet-rich plasma; M,
medium concentration platelet-rich plasma; H, high concentration
platelet-rich plasma; C, collagen gel; *, statistically significant, p < 0.05).
968 M. KAWASUMI, H. KITOH, K. A. SIWICKA, N. ISHIGURO
THE JOURNAL OF BONE AND JOINT SURGERY
320 g and 380 g was followed by an osteotomy at the level
of the diaphysis.20 Weight-bearing as tolerated was allowed
immediately after the operation. Seven days after operation
lengthening was initiated at a rate of 0.375 mm twice daily
for ten days (7.5 cm of total distraction). Immediately after
the completion of lengthening, 150 μl of each type of gel
containing rat bone-marrow cells at a density of 1 × 107
cells/ml were injected into the distraction callus under fluo-
roscopic guidance. The rats were divided into five groups,
according to the type of injected gel as follows: group P
(n = 16), group L (n = 20), group M (n = 20), group H
(n = 20), and group C (n = 15).
Radiological evaluation. This was performed using a soft
radiograph apparatus (Softex ES/M; Softex Co, Tokyo,
Japan). Lateral radiographs were taken at one, two, and
four weeks after injection of gel. They were evaluated using
image-analysis software (Scion Image for Windows, Scion
Co, Frederick, Maryland). The distraction gap was outlined
as a quadrilateral region of interest from the outside corners
of the two proximal and the two distal cortices. Mineralised
new bone was defined as any region with a density equiva-
lent to or greater than the adjacent medullary bone.
In vivo micro-CT. Representative specimens of distracted
femora (six rats in each group, 30 in total) were examined
using a high-resolution micro-CT imaging system (ScanX-
mate-A100S; Comscantecno Co. Ltd, Kanagawa, Japan)
four weeks after injection of bone-marrow cells.
Volumetric measurements of mineralised new bone were
made using 3-D image analysis software (TR1/3D-BON;
Ratoc System Engineering Co. Ltd, Tokyo, Japan). The vol-
ume of the distraction gap was also recorded and it repre-
sented the volume of interest which was mineralised.
Statistical analysis. Equality of variances was verified using
the Bartlett test (Stat View for Windows version 5.0; SAS
Institute Japan Ltd, Tokyo, Japan). One-way analysis of
variance was used for comparison of groups, supported by
the Bonferroni-type multiple comparison. Statistical signif-
icance was set at a p-value ≤ 0.05. All the results are
expressed as the mean and SEM (±).
Properties of platelet-rich plasma and platelet-poor plasma.
The mean concentration of platelets (platelets/μl) was
4358 ± 265 × 103 in high concentration, 1453 ± 88 × 103 in
medium concentration, 48 ± 29 × 103 in low concentration,
8 ± 2 × 103 in platelet-poor plasma and 413 ± 54 × 103 in
whole blood. The mean concentration of platelets in the
low, medium and high concentration groups was 117%
(76% to 146%), 352% (263% to 441%) and 1055%
(695% to 1233%) of that in whole blood, respectively.
There was a ninefold difference in the mean platelet con-
centration between the low and high concentration groups.
There was no significant difference in the concentration of
fibrinogen among the groups.
In vitro cell proliferation. Group H showed a significant
increase in the proliferation of rat bone-marrow cells com-
pared with the remaining groups on days 2, 4 and 6 (analysis
of variance (ANOVA), p < 0.05). By contrast, groups L and M
did not show increased cell proliferation, compared with
group P (Fig. 1).
In vitro osteogenic differentiation. On day 6, the alkaline
phosphatase activity was significantly increased in group C
(ANOVA, p < 0.05), compared with the other groups (Fig.
2a). Group P L, M and H showed no significant increase in
Day of culture
Alkaline phosphatase activity (mmol/P)
Graphs showing a) the concentration of alkaline phosphatase in the cell lysates and b) alkaline-phosphatase expression as measured by
mRNA in rat bone-marrow cells cultured with platelet-rich plasma gels of different platelet concentrations and collagen gel. The alkaline phos-
phatase activity was significantly increased in group C on day six and there was also significantly enhanced expression of alkaline phos-
phatase mRNA in this group. (P, platelet-poor plasma; L, low concentration platelet-rich plasma; M, medium concentration platelet-rich
plasma; H, high concentration platelet-rich plasma; C, collagen gel; *, statistically significant, p < 0.05).
Day of culture
THE EFFECT OF THE PLATELET CONCENTRATION IN PLATELET-RICH PLASMA GEL ON THE REGENERATION OF BONE969
VOL. 90-B, No. 7, JULY 2008
alkaline phosphatase activity. Similarly, the expression of
alkaline phosphatase mRNA was significantly increased in
group C (ANOVA, p < 0.05) while there was no significant
differences in the other groups (Fig. 2b).
Radiological evaluation. The formation of callus was
enhanced in a platelet dose-dependent manner among the
scaffolds. At four weeks after completion of distraction,
radiological union was evident in group H only. In the
Plain radiographs of lengthened femora of rats after injection of rat bone-marrow cells embedded in
platelet-rich plasma gel with different platelet concentrations or collagen gel into the distraction group.
Radiographs were taken at one, two, and four weeks after transplantation. Complete bridging of the gap
was observed in group H at four weeks (P, platelet-poor plasma; L, low concentration platelet-rich
plasma; M, medium concentration platelet-rich plasma; H, high concentration platelet-rich plasma; C, col-
970 M. KAWASUMI, H. KITOH, K. A. SIWICKA, N. ISHIGURO
THE JOURNAL OF BONE AND JOINT SURGERY
remaining groups, the central radiolucent area (fibrous
zone) was still present (Fig. 3). In group H, satisfactory for-
mation of callus was seen soon after the injection of the rat
bone-marrow cells, resulting in an early bridging of the dis-
Significantly larger areas of mineralised bone were found
in group H than in the other groups at four weeks after
injection of the rat bone-marrow cells (ANOVA, p < 0.05).
There were no significant differences in bone formation
among groups P, L, M, and C (Fig. 4).
Micro CT. Favourable bone formation was observed in
groups H and C (Fig. 5). Quantitative 3-D CT showed that
the volume of the distraction gap was largest in group H,
although there were no statistically significant differences
among any group (Fig. 6).
We have previously shown a satisfactory outcome in fem-
oral lengthening enhanced by transplantation of culture-
expanded bone-marrow cells and platelet-rich plasma into
the callus.15 However, the beneficial effect of cell therapy on
osteogenesis was less pronounced at the anteromedial aspect
of the tibia.16,17 To improve our bone-marrow cells and pla-
tet-rich plasma cell therapy, it was necessary to determine
the concentration of platelets in the plasma gel which would
give the optimal proliferation and differentiation capability
of the bone-marrow cells. In our study, the 3-D culture sys-
tem was used to analyse the proliferation and differentiation
of rat bone-marrow cells in platelet-rich plasma gels con-
taining variable concentrations of platelets. In addition, the
effect of transplantation of bone-marrow cells on distrac-
tion osteogenesis was evaluated in vivo in the rat limb-
lengthening model. To the best of our knowledge, this is the
first report which has investigated the quality of regenerate
bone during distraction osteogenesis after transplantation
of rat bone-marrow cells combined with variable concentra-
tions of platelets in platelet-rich plasma gel.
Platelet-rich plasma with a higher platelet concentration
significantly enhanced the proliferation of rat bone-
marrow cells, although the rate of differentiation of osteo-
blasts was not accelerated. However, the effect of platelet-
rich plasma on the proliferation and differentiation of
osteoblastic cells is still controversial. Choi et al,8 using
canine platelet-rich plasma and alveolar bone cells, showed
that the viability and proliferation of the latter were sup-
pressed by high, but were stimulated by low concentrations
of platelet-rich plasma. Kanno et al23 noted that human
platelet-rich plasma inhibited activity in the osteoblastic
cell line during the growth phase, but stimulated it when
the cells attained confluence. Graziani et al9 showed that
moderate platelet concentrations in human platelet-rich
plasma stimulated the differentiation of human osteoblasts.
Lucarelli et al10 stated that 10% platelet-rich plasma pro-
moted the proliferation of stromal stem cells derived from
human bone marrow. The results of previous studies may
differ from our findings because of the different origins of
platelet-rich plasma used or cell types examined. The
amount of platelet-rich-plasma-derived growth factors var-
ied depending on the animal species,24 and the cellular
response to these growth factors may also depend on the
phenotype of the cells tested. However, similar results were
reported by Arpornmaeklong et al11 and Ogino et al,12 who
analysed the effect of platelet concentrations in rat platelet-
rich plasma on the proliferation and differentiation of rat
bone-marrow cells. Arpornmaeklong et al11 noted that
platelet-rich plasma caused a dose-dependent stimulation
of cell proliferation while reducing alkaline phosphatase
activity and calcium deposition in the 3-D culture. Ogino et
al12 showed that platelet-rich plasma stimulated the prolif-
eration but suppressed the differentiation in the monolayer
culture system. These studies suggested that platelet-rich
plasma could have a beneficial effect on the proliferation of
bone-marrow cells, without promoting osteoblastic differ-
The quality of the regenerated bone was significantly
improved after transplantation of rat bone-marrow cells
with a plasma with a higher platelet concentration,
although these cells showed no increase in alkaline phos-
phatase activity in the in vitro 3-D culture, compared with
gels with a lower concentration of platelets. We have previ-
ously observed elevated levels of alkaline phosphatase
activity in rat P2 cells cultured with differentiation medium
containing dexamethasone.22 In the present study, the
embedded rat P2 bone-marrow cells used for transplanta-
tion could have differentiated into an osteoblastic pheno-
type during the monolayer culture before transplantation
into the callus. The positive effect on osteogenesis in group
H may have resulted from the increased potential for prolif-
eration of the osteoblastic cells within the gels.
Bone area (mm2)
Graph showing the quantification of mineralised bone area in the dis-
traction callus. Significantly enhanced bone formation was observed in
group H (ANOVA, p < 0.05) (P, platelet-poor plasma; L, low concentra-
tion platelet-rich plasma; M, medium concentration platelet-rich plasma;
H, high concentration platelet-rich plasma; C, collagen gel).
THE EFFECT OF THE PLATELET CONCENTRATION IN PLATELET-RICH PLASMA GEL ON THE REGENERATION OF BONE971
VOL. 90-B, No. 7, JULY 2008
Rat bone-marrow cells embedded in collagen gels showed
the most favourable effect on osteogenic differentiation in
vitro, and they also resulted in good bone formation in vivo.
Type-I collagen, as a carrier, is considered to maintain the
phenotype of osteoblasts, and the combination of cultured
osteoblasts and type-I collagen is commonly-used in tissue
engineering.25,26 We have previously demonstrated that
culture-expanded bone-marrow cells mixed with collagen
gel accelerated the maturation of regenerate bone and short-
ened the consolidation period during distraction osteo-
genesis in rats.20 Type-I collagen is, however, typically
obtained from bovine hides and has the associated risks of
immune reaction and disease transmission. On the other
hand, autologous platelet-rich plasma is non-toxic and non-
immunoreactive and appears to be safe for clinical use. Bone-
marrow cells with a high concentration of platelet-rich
plasma showed lower alkaline phosphatase activity and
mRNA expression compared with those with collagen gels,
but transplantation of bone-marrow cells and high concen-
tration platelet-rich plasma significantly improved the radio-
logical appearance of regenerate bone. Quantitative micro-
CT showed the largest bone volume within the distraction
gap of group H, but the small sample sizes precluded the
determination of any statistically significant difference
between groups C and H. Therefore, a platelet-rich plasma
with a higher concentration of platelet could be a safe substi-
tute for collagen scaffolds for bone-marrow cell therapy in
In conclusion, a higher concentration of platelets in the
platelet-rich plasma gel stimulated the proliferation of rat
bone-marrow cells, but did not promote osteoblastic differ-
entiations. Rat bone-marrow cells with a higher concentra-
tion of platelets in the platelet-rich plasma had the most
favourable effect on osteogenesis in the rat limb-lengthening
model. Our results indicate that in clinical practice platelet-
rich plasma with a high concentration of platelets may be a
useful adjunct for bone regeneration during distraction
No benefits in any form have been received or will be received from a commer-
cial party related directly or indirectly to the subject of this article.
Micro-CT scans of the distraction gap at four weeks after cell transplantation. Satisfactory bone formation was demonstrated in groups H and C (P,
platelet-poor plasma; L, low concentration platelet-rich plasma; M, medium concentration platelet-rich plasma; H, high concentration platelet-rich
plasma; C, collagen gel).
Bone volume (mm3)
Graph showing quantitative volumetric analysis of bone regenerates.
The largest bone volume was observed in group H (P, platelet-poor
plasma; L, low concentration platelet-rich plasma; M, medium concen-
tration platelet-rich plasma; H, high concentration platelet-rich plasma;
C, collagen gel).
972M. KAWASUMI, H. KITOH, K. A. SIWICKA, N. ISHIGURO Download full-text
THE JOURNAL OF BONE AND JOINT SURGERY
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