Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569

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Journal of Oral and Maxillofacial Surgery,

Medicine, and Pathology
journal homepage: www.elsevier.com/locate/jomsmp

Oral Medicine/Original research

Comparison study between plasma rich in growth factors and

platelet-rich plasma for osteoconduction in rat calvaria
Takashi Eda ∗ , Kosuke Takahashi, Shingo Kanao, Akinobu Aoki, Naomi Ogura, Ko Ito,
Hiroyasu Tsukahara, Masaaki Suemitsu, Kayo Kuyama, Toshirou Kondoh
Department of Maxillofacial Surgery, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Plasma rich in growth factors (PRGF) and platelet-rich plasma (PRP) can be rapidly obtained
Received 22 January 2017 from patient blood. They are a new and potentially useful adjunct in oral and maxillofacial bone repair or
Received in revised form 9 April 2017 regenerative surgery. The aim of this study was to compare the possibility of new bone formation using
Accepted 28 June 2017
PRGF and PRP.
Available online 13 July 2017
Methods: The osteogenic potential with transplantation of PRGF or PRP onto rat calvaria was evaluated
by histologic examination and microCT. PRGF or PRP was prepared by centrifugation of rat whole blood
Keywords:
(WB). First, the cells in the blood product were counted; there were no leukocytes in PRGF, and PRP
Plasma rich in growth factors
Platelet-rich plasma
included leukocytes. PRGF contained higher levels of TGF-␤1 and PDGF-BB than PRP. Furthermore, PRGF
Bone regeneration or PRP was transplanted onto calvarial bone of rats.
Results: MicroCT showed that PRGF promoted an increase in bone volume when compared to PRP. His-
tological observation demonstrated that the PRGF group showed newly formed bone in a wide range. In
addition, the PRP group showed numerous inflammatory cells compared to the PRGF group in HE-stained
specimens. This suggests that PRP might delay bone regeneration due to the inflammatory response.
Conclusions: PRGF has more availability for bone regeneration than PRP, and PRGF may be useful in bone
regeneration treatment.
© 2017 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽

1. Introduction to have several advantages, including the enhancement of bone

Platelet concentration products, which are autologous con-
stituents of inductive factors obtained from blood, have high
concentrations of platelets containing various growth factors [1].
The concentration of autologous platelets in plasma [platelet-rich
plasma (PRP)] and the growth factors contained within platelets
have been studied since 1998 [2]. The previous study reported that
the combined use of autogenous bone with PRP increased radio-
graphic and histomorphometric bone densities [2]. In addition, PRP
has been used in bone augmentation for dental implants or frac-
ture healing of jaw bone [2–5]. However, it has been shown that
PRP formulations have different biological activities, depending on
their preparation and administration [6–8].
The preparation of plasma rich in growth factors (PRGF) is one
way to concentrate platelets [9,10], and in 1999, it was shown
regeneration and rapid soft tissue healing [11]. This system is
advantageous as it requires only one step of centrifugation and
is leukocyte-free, thus avoiding higher levels of pro-inflammatory
cytokines [3]. In addition, PRGF contains high levels of growth
factors such as transforming growth factor (TGF)-␤ and platelet-
derived growth factor (PDGF), which are associated with bone
regeneration [12]. However, there has been little basic research into
the efficacy of PRGF in bone regeneration [9].
The aim of this study was to compare between PRGF and PRP
to the availability of bone formation in transplantation of using
histologic findings and microCT analysis.
2. Materials and methods
2.1. Preparations of PRGF and PRP

夽 AsianAOMS: Asian Association of Oral and Maxillofacial Surgeons; ASOMP: Asian
Society of Oral and Maxillofacial Pathology; JSOP: Japanese Society of Oral Pathol-
ogy; JSOMS: Japanese Society of Oral and Maxillofacial Surgeons; JSOM: Japanese
Society of Oral Medicine; JAMI: Japanese Academy of Maxillofacial Implants.
∗ Corresponding author.
E-mail address: Eda.takashi.12.22@gmail.com (T. Eda).
A total of 27 Sprague-Dawley male rats (age: 15 weeks) weigh-
ing 405–415 g were purchased from Japan SLC (Shizuoka, Japan).
Rat whole blood (5 ml) was collected from the external jugular
vein by syringe aspiration via direct venipuncture with a 21-gauge
needle. Blood was immediately placed into 5-ml sterile extraction

http://dx.doi.org/10.1016/j.ajoms.2017.06.011
2212-5558/© 2017 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd. All rights reserved.夽
564 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569

Fig. 1. Blood plasma fractions were obtained from citrate-anticoagulated whole blood.
(A) Tubes were centrifuged at 460g for 8 min to give PRGF. The bottom fraction contains erythrocytes, and the top fraction contains platelets. Both fractions are separated
by the whitish buffy coat, which contains leukocytes. The plasma fraction (1 ml over the buffy coat) was collected to prepare Fraction 2 (F2). Fraction 1 (F1) was prepared
from the upper layer of F2. (B, C) PRP was prepared at a separation spin of 1000 × g for 4 min, followed by a concentration spin of 800 × g for 9 min. (B) The layer of plasma
containing the buffy coat was concentrated. (C) Plasma (500 ␮l) containing WBCs and RBCs were collected to prepare PRP. PPP was prepared from the upper layer of PRP in
plasma.

tubes containing 0.5 ml of 3.8% sodium citrate as an anticoagulant
(BTI Biotechnology Institute, Vitoria, Spain).
PRGF was prepared from whole blood (WB) centrifuged in accor-
dance with Anitua’s protocol [9,10,13] (Fig. 1A). Briefly, tubes were
centrifuged at 460g for 8 min. The bottom fraction containing ery-
throcytes and the top fraction containing platelets are separated
by the whitish buffy coat, which contains leukocytes. The plasma
fraction (1 ml over the buffy coat) was collected as Fraction 2 (F2),
while Fraction 1 (F1) was the layer above F2.
PRP was prepared from WB using the double-spin method
described by Marx [5] (Fig. 1B and C). Briefly, WB was centrifuged
at 1000 × g for 4 min, and the plasma layer containing the buffy
coat was collected (Fig. 1B), followed by centrifugation at 800g for
9 min. The lower plasma layer (500 ␮l) containing white blood cells
(WBCs) and red blood cells (RBCs) was collected to prepare PRP. The
upper layer was platelet-poor plasma (PPP) (Fig. 1C). All animals
were maintained and used in accordance with the Nihon University
Intramural Animal Use guidelines (Ethics Committee Registration
No: AP12MD018).
2.2. Characteristics of autologous blood product
Platelets, WBCs, and RBCs in PRGF and PRP were counted using
Celltac ␣ (MEK-6358; NIHON KOHDEN, Tokyo, Japan). Samples of
serum, PPP, PRP, PRGF F1, and PRGF F2 for evaluating growth factor
concentrations were stored at −80 ◦ C before use. Levels of TGF-␤1,
PDGF-BB, and insulin-like growth factor (IGF)-1 were measured
using ELISA (enzyme-linked immunosorbent assay) kits (Quan-
tikine colorimetric ELISA kits; R&D Systems, Minneapolis, MN) in
accordance with the manufacturer’s instructions.
2.3. Transplantation procedure
The transplantation procedure was designed to evaluate newly
formed bone in the presence of PRP or PRGF F2 with artificial dam
preparation using PTFE tubes (inner diameter, 3 mm; outer diame-
ter, 4 mm; height, 2 mm) in vivo. Rats were randomly divided into
three groups (n = 3 per group): the Control group (PTFE tube only),
the PRP group (PRP in the PTFE tube), and the PRGF group (PRGF F2
in the PTFE tube).
All animals were anesthetized by intraperitoneal injection of
®
25 mg/kg pentobarbital sodium (Somnopentyl ; Kyoritsu Seiyaku,
Tokyo, Japan), and the surgical site was prepared aseptically. A
previous study reported that PRGF F2 had a higher platelet con-
centration and growth factors compared with PRGF F1 [9,13]. From
this kind of reason, we used F2 in transplantation. Platelets in PRP
or PRGF F2 were activated using 10% calcium chloride solution just
prior to use [9,14,15]. Transplantation protocol into rats was per-
formed as in previous reports [16–18]. Briefly, a linear skin incision
was made along the edge of the skull with a #15 surgical blade.
The periosteum of the calvaria was carefully removed with a ras-
patorium after elevation of the skin-periosteum flap. PRP or PRGF
F2 was inserted into PTFE tubes, which were then placed on the
calvarial surface. Overlying tissue was closed with sutures.
2.4. MicroCT analysis of transplants
Quantitative image analysis of new bone formation was per-
formed using an in vivo microCT system (Rigaku-mCT, Tokyo,
Japan). Rats were deeply anesthetized with intraperitoneal injec-
tion of sodium pentobarbital (25 mg/kg) and individually placed on
the object stage. Calvarial bone at the PTFE tube site was scanned
using a microCT system with an X-ray source of 90 kV/150 ␮A at
2, 4, and 8 weeks after transplantation. Imaging of newly formed
bone in the area containing the inner cavity of the PTFE tube was
then performed over a full 360◦ , with an exposure time of 2 min.
An isotropic resolution of 20 × 20 × 20 ␮m3 voxels was selected,
which displayed the microstructure of new bone formation in rats.
The original 3D images were displayed and analyzed using I-view
software (J. Morita, Kyoto, Japan). Analysis of new bone formation
was performed in the area containing the inner cavity of the PTFE
tube. Linearity of the micro-CT scanner was established by scanning
 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569
Fig. 2. Counting of inflammatory cells.
(A) The degree of inflammation is objectively assessed using ImageJ in terms of number of inflammatory cells, such as plasma cells and lymphocytes per unit area
(500 × 500 ␮m) in the tube center by H-E staining at 2 weeks after surgery. Columns represent mean ± standard deviation. ** P < 0.01. (B-D) Inflammatory cells are observed
by H-E staining at 2 weeks after surgery. The control group (B), the PRGF group (C) and the PRP group (D). Original magnification × 200.
a phantom containing several densities of a standard calibration
material. The 3D image of newly formed bone was measured, and
structural indices of the newly formed bone were calculated using
a morphometric program (TRI/3D-BON; Ratoc System Engineering,
Tokyo, Japan). 3D analysis directly measured bone mineral den-
sity (BMD = BMC/BV; mg/cm3 ), bone mineral content (BMC; mg),
and bone volume (BV; cm3 ) of the new bone formation in the cav-
ity of the PTFE tube. In this experiment, a circle with a radius of
1.5 mm defined to a height of 2.0 mm from the parietal bone to the
periosteum along PTFE tubes as the ROI of bone mineral quantity
analysis.
2.5. Histologic analysis
Rats were sacrificed 2, 4, and 8 weeks after transplantation.
The calvarial bone area including the PTFE tube was extirpated
en bloc. Samples were fixed in 10% neutral-buffered formalin solu-
tion, decalcified in 10% EDTA (pH 7.0), and embedded in paraffin.
Semi-serial sections (4 ␮m) were obtained in the sagittal orienta-
tion. Sections were stained with hematoxylin and eosin (H-E) and
observed by light microscopy. Newly formed bone was defined as
bone-like hard tissue arranged regularly with osteoblasts between
the cortical bone and periosteum. Inflammatory changes were
observed in each group at 2 weeks after surgery in H-E-stained
specimens. At that time, infiltration of inflammatory cells was
observed, and the density of inflammatory cells, such as plasma
cells and lymphocytes, in the tissue was elevated. The degree of
inflammation was objectively assessed using ImageJ in terms of
the number of inflammatory cells per unit area [19,20]. The origi-
nal file type of the photographs was Tagged Image File Format (TIFF)
with a size of 4080 × 3072 pixels (872 × 656 ␮m). Cell counting was
performed (500 ␮m2 ) in the tube center under high magnifica-
tion (200 × ) (Fig. 2B-D). Images were binarized with thresholds.
Nucleus counting was selected depending on pixels (pixel size
250–500) and circularity (0.80–1.00). The number of cell nuclei
was considered to be equivalent to the number of inflammatory
cells (Fig. 2A).
2.6. Immunohistochemical analysis
Sections of calvarial bone including the PTFE tube from
all groups were stained immunohistochemically. Sections were
deparaffinized, and endogenous peroxidase activity was quenched
by incubation in 3% H2 O2 in methanol for 15 min at room temper-
ature. Sections were washed with PBS (phosphate-buffered saline)
and incubated with goat polyclonal antibody against rat Runx2
(1:50; sc-12488; SANTA CRUZ, Tokyo, Japan), rabbit polyclonal
antibody against rat osterix (1:100, ab22552; Abcam, Tokyo, Japan),
and rabbit polyclonal antibody against rat osteocalcin (1:100,
565
Table 1
Cell count of platelet, white blood cell, and red blood cell in plasma preparations
(n = 3).
Sample Platelets ( × 104 /␮l) WBCs ( × 103 /␮l) RBCs ( × 104 /␮l)
WB 97.7 ± 1.9 5.3 ± 0.1 908.0 ± 10.0
F1 44.0 ± 2.6 0 4.6 ± 1.6
F2 241.4 ± 19.2 0 84.6 ± 1.6
PPP 0.7 ± 0.4 0 0.8 ± 0.2
PRP 361.2 ± 34.0 2.8 ± 2.3 28.5 ± 8.6
(WB; whole blood, F1; PRGF Fraction 1, F2; Fraction 2, PPP; platelet-poor plasma,
PRP; platelet-rich plasma, WBCs; white blood cells, RBCs; red blood cells).
LB4005;Cosmo Bio, Tokyo, Japan) for 1 h at room temperature.
Secondary antibody staining was performed with reagents from
ChemMate ENVISION kit/HRP (DAB; Dako, Glostrup, Denmark), in
accordance with the manufacturer’s protocol. Immune complexes
were visualized using 3 3 -diaminobenzidine tetrachloride (DAB;
Merck, Darmstadt, Germany). Immunostained sections were then
counterstained with Mayer’s hematoxylin. Normal bone was used
as a positive control in immunochemical staining, and negative
controls were prepared by omitting primary antibody [21].
2.7. Statistical analysis
All results are expressed as the mean ± standard deviation (SD).
The ANOVA test was used to compare three groups. Tukey’s test
followed the ANOVA test. The significance of differences was evalu-
ated by Tukey’s test under a normal distribution and equal variance.
Differences were considered significant at P < 0.05.
3. Results
3.1. Characteristics of autologous blood products
Concentrations of platelets, WBCs, and RBCs in each sample are
shown in Table 1. Platelet concentration was 2.5-fold higher in PRGF
F2 and 3.7-fold higher in PRP, as compared to WB. Platelets were not
concentrated in PPP and PRGF F1 when compared to WB. No WBCs
were present in PRGF F1, PRGF F2, or PPP, while PRP included WBCs.
Furthermore, all blood products contained RBCs.
The concentrations of TGF-␤1, PDGF-BB, and IGF-1 were then
evaluated in each blood product. PRGF F1 and PRGF F2 contained
higher levels of TGF-␤1, PDGF-BB, and IGF-1 when compared with
serum, PPP, and PRP (Table 2).
3.2. MicroCT analysis
MicroCT was used to quantify the mineralization within the
PTFE tubes at 2, 4, and 8 weeks, allowing assessment of the entire
566 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569

Fig. 3. Color images showing bone mineral density by TRI/3D-BON.

Color images showed bone mineral density at (A-C) 2, (D-F) 4 and (G-I) 8 weeks; the control group (A, D, G), the PRGF group (B, E, H) and the PRP group (C, F, I). (B) The inner
side of the tube shows a small amount of newly formed bone cultured with PRGF at 2 weeks. (D–I) The area of new bone formation has progressed into the tube in all groups
at 4 and 8 weeks. Scale bar; 3000 ␮m.

Table 2 than in the PRP group at 2, 4, and 8 weeks (Fig. 4 D-F). Similarly,
Levels of growth factors in plasma preparations (n = 3).
the value for BV was higher in the PRGF group than in the control
Sample TGF-␤1 (ng/ml) PDGF-BB (ng/ml) IGF-1 (ng/ml) group at 8 weeks (Fig. 4F), although the values for BV did not differ
Serum N.D. N.D. 1187.50 ± 94.60 in the PRGF group when compared to the control group at 2 and 4
F1 31.35 ± 0.57 1.04 ± 0.31 1387.50 ± 47.80 weeks (Fig. 4D and E).
F2 115.80 ± 2.13 5.01 ± 0.48 1237.50 ± 125.00
PPP 5.67 ± 0.07 N.D. 1000.00 ± 40.00
PRP 7.40 ± 0.35 N.D. 876.67 ± 25.17 3.3. Histological findings
(WB; whole blood, F1; PRGF Fraction 1, F2; Fraction 2, PPP; platelet-poor plasma,
PRP; platelet-rich plasma, TGF; transforming growth factor, PDGF; platelet-derived Newly formed bone was observed between the periosteum and
growth factor, IGF; insulin-like growth factor). cortical bone in the PRGF group at 2 weeks after transplantation
(Fig. 5B). However, little newly formed bone was observed in the
PRP group and the control group (Fig. 5A and C). As compared to the
volume of the new bone. Fig. 3 shows BMD color images of trans-
other groups, the PRGF group showed a wide range of newly formed
plants with microCT analysis. The inner PTFE tube contained a small
bone at 4 and 8 weeks after surgery (Fig. 5E and H). In addition,
amount of newly formed bone on the calvarial side at 2 weeks
osteoblasts were observed around the cortical and newly formed
(Fig. 3A-C). A small volume of newly formed bone was seen in
bone (data not shown). Next, the number of inflammatory cells was
the PRGF group when compared with the control and PRP groups
counted in H-E-stained sections at 2 weeks after transplantation
(Fig. 3A-C). The newly formed bone area increased inside the PTFE
(Fig. 2B-D). The PRP group showed numerous inflammatory cells,
tube after 4 and 8 weeks, as compared to after 2 weeks (Fig. 3D-
such as plasma cells, when compared to the other groups in the
I). In addition, the newly formed bone area increased inside the
tube center at 2 weeks (Fig. 2A).
PTFE tube in the PRGF group when compared to the control and
PRP groups (Fig. 3B, E and H). Furthermore, the PRGF group mostly
showed newly formed bone in the PTFE tube center area at 8 weeks 3.4. Immunohistological findings
(Fig. 3H).
BMD and BV were then analyzed using microCT (Fig. 4). BMD Immunohistochemical findings for Runx2, osterix, and osteocal-
did not differ among the three experimental groups at 2, 4, and 8 cin are shown in Fig. 6. Osteoblasts positive for Runx2 and osterix
weeks (Fig. 4A-C). The values for BV was higher in the PRGF group were seen in the margin of newly formed bone in all experimental
 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569 567

Fig. 4. Quantitative measurement of BMD and BV.

BMD (A) and BV (D) at 2 weeks, BMD (B) and BV (E) at 4 weeks, and BMD (C) and BV (F) at 8 weeks. (BMD; bone mineral density, BV; bone volume. Columns represent
mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.005).

Fig. 5. Histologic findings.

H-E staining showed new bone formation at 2 (A-C), 4 (D-F) and 8 weeks (G-I); the control group (A, D, G), the PRGF group (B, E, H) and the PRP group (C, F, I). (A-C) New bone
formation is observed on the calvarial bone side at 2 weeks. (B) New bone formation is observed in the PRGF group at 2 weeks. (A, C) Little newly formed bone is observed in
the PRP group and the control group. (D–I) A wide range of newly formed bone formation is observed at 4 and 8 weeks. Original magnification × 40. Scale bar; 500 ␮m. NB;
new bone, CB; calvarial bone, T; tube.

groups, and positive osteocytes were seen around newly formed Runx2 (Fig. 6A-C) and osterix (Fig. 6D-F). Osteocalcin (Fig. 6G-I)
bone in the PRGF group at 2 weeks (Fig. 6A-F). Numerous swollen staining was also seen in osteoblasts arranged in the margin and in
spindle-shaped cells in the connective tissue were positive for osteocytes around newly formed bone in the PRGF group at 2 weeks
568 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569

Fig. 6. Representative immunohistochemistry.

Runx2 (A–C), osterix (D–F), and osteocalcin (G–I) at 2 weeks. the control group (A, D, G), the PRGF group (B, E, H), and the PRP group (C, F, I). Original magnification × 400.
Scale bar; 100 ␮m. NB; new bone, CB; calvarial bone. Arrows indicate positive reactions.

(Fig. 6H). However, osteocalcin staining was negative in the control phenomenon when bone resorption and formation are occurring
and PRP groups at 2 weeks (Fig. 6G and I). In addition, immunohis- simultaneously [28,29]. This suggests that the control and PRP
tochemical staining for Runx2, osterix, and osteocalcin was positive groups at 2 weeks showed no osteocalcin because bone calcification
in all groups at 4 and 8 weeks (data not shown). had not started. In other hands, PRGF groups at 2 weeks accelerate
osteogenic differentiation and bone calcification.
This study showed that PRGF F1 and F2 contained higher lev-
4. Discussion
els of TGF-␤1, PDGF-BB, and IGF-1 than PRP (Table 2). It is known
that TGF-␤1 plays an important role in bone metabolism and in
In this study, bone formation was compared between PRGF and
the early phase of bone regeneration and remodeling [30]. TGF-␤
PRP transplanted onto rat calvaria. The grafting of PRGF accelerated
stimulated osteoblast differentiation from mesenchymal stem cells
new bone formation more than PRP. New bone formation at the
from bone marrow [31,32]. In vivo studies have shown that a lack of
grafting sites in the PRGF group was observed earlier and in greater
TGF-ß1 leads to a weaker bone structure and less mechanical sta-
amounts than in the PRP and control groups on microCT analysis
bility [33]. PDGF was confirmed to have a positive effect on bone
and histological observation (Figs. 3 and 5).
healing, and there were generally higher concentrations of PDGF
Immunohistological observations showed that Runx2, osterix,
observed during bone repair [34,35]. IGF-I is one of the most abun-
and osteocalcin were expressed in osteocytes around the newly
dant growth factors in the human skeleton [36] and is thought to
formed bone (Fig. 6). Runx2 and osterix, which are transcription fac-
be important for both systemic and local functions in skeletal and
tors that are critical for bone formation [22–24]. In addition, osterix
bone metabolism [37]. A higher concentration of these growth fac-
acts downstream of Runx2 during the differentiation of pluripotent
tors in PRGF appears to be one of the advantageous factors for new
mesenchymal cells into the osteoblastic lineage [24–26]. Runx2
bone formation.
and osterix were detected in all experimental groups. However,
PRP had a higher platelet concentration than PRGF and included
Osteocalcin was detected in the PRGF groups earlier than in the
WBCs in this study. A previous study reported that PRP had a
PRP groups. Osteocalcin was positive in the PRGF group, whereas
high platelet concentration when compared with PRGF in humans
the control and PRP groups were negative, at 2 weeks after trans-
[2,9,10]. In addition, the PRP group showed numerous inflamma-
plantation. Osteocalcin is known to be involved in osteogenic
tory cells, such as plasma cells and WBCs, when compared with
differentiation and is secreted by osteoblasts after bone calcifica-
the other groups at 2 weeks. Some researchers have suggested that
tion starts [27]. Osteocalcin is also associated with the turnover
 T. Eda et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 29 (2017) 563–569
platelets play a role in the recruitment of inflammatory cells such as
neutrophils and monocytes [4,38], and platelets themselves act as
innate inflammatory cells with acute host defense function [4,38].
Furthermore, a significant increase in IL-1␤ was observed from PRP
when the concentration of WBCs was large, thus suggesting that
IL-1␤ synthesis from PRP is related to WBCs [39,40]. In comparison
with PRGF and PRP, PRP was considered to induce inflammatory
reactions based on the relationship with WBCs, platelet number,
and cytokines released, and it is suggested that PRP is undesirable
for treatment of bone regeneration therapy for these reason.
5. Conclusions
In conclusion, microCT and histological observation demon-
strated that PRGF enhanced new bone formation from early time
points compared to PRP. In addition, PRP delays new bone for-
mation due to the inflammatory response. Thus, it appears that
PRGF can enhance bone regeneration and may be available in bone
regeneration treatment.
Role of the funding source
No funding was received for this study.
Acknowledgment
The authors have no conflicts of interest to declare.
References
[1] Park SY, Kim KH, Gwak EH, Rhee SH, Lee JC, Shin SY, et al. Ex vivo bone
morphogenetic protein 2 gene delivery using periodontal ligament stem cells
for enhanced re-osseointegration in the regenerative treatment of
peri-implantitis. J Biomed Mater Res A 2014;103:38–47.
[2] 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;85:638–46.
[3] Dohan Ehrenfest DM, Bielecki T, Del Corso M, Inchingolo F, Sammartino G.
Shedding light in the controversial terminology for platelet-rich products:
platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet-leukocyte gel
(PLG), preparation rich in growth factors (PRGF), classification and
commercialism. J Biomed Mater Res A 2010;95:1280–2.
[4] Malhotra A, Pelletier MH, Yu Y, Walsh WR. Can platelet-rich plasma (PRP)
improve bone healing: a comparison between the theory and experimental
outcomes. Arch Orthop Trauma Surg 2013;133:153–65.
[5] Mark RE, Garg AK. The science of platelet-rich plasma. In: Lisa CB, editor.
Dental and craniofacial applications of platelet-rich plasma. Chicago:
Quintessence Publishing; 2005. p. 33–9.
[6] Aghaloo TL, Moy PK, Freymiller EG. Investigation of platelet-rich plasma in
rabbit cranial defects: a pilot study. J Oral Maxillofac Surg 2002;60:1176–81.
[7] Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet-rich plasma on bone
regeneration in autogenous bone graft. Int J Oral Maxillofac Surg
2004;33:56–9.
[8] Gerard D, Carlson ER, Gotcher JE, Jacobs M. Effects of platelet-rich plasma on
the healing of autologous bone grafted mandibular defects in dogs. J Oral
Maxillofac Surg 2006;64:443–51.
[9] Anitua E, Tejero R, Zalduendo MM, Orive G. Plasma rich in growth factors
promotes bone tissue regeneration by stimulating proliferation, migration,
and autocrine secretion in primary human osteoblasts. J Periodontol
2013;84:1180–90.
[10] Anitua E, Troya M, Orive G. An autologous platelet-rich plasma stimulates
periodontal ligament regeneration. J Periodontol 2013;84:1556–66.
[11] Anitua E. Plasma rich in growth factors: preliminary results of use in the
preparation of future sites for implants. Int J Oral Maxillofac Implants
1999;14:529–35.
[12] Anitua E, Begoňa L, Orive G. Controlled ridge expansion using a two-stage
split-crest technique with ultrasonic bone surgery. Implant Dent
2012;21:163–70.
[13] Yoshioka T, Kanamori A, Washio T, Aoto K, Uemura K, Sakane M, et al. The
effects of plasma rich in growth factors (PRGF-Endoret) on healing of medial
569
collateral ligament of the knee. Knee Surg Sports Traumatol Arthrosc
2013;21:1763–9.
[14] Dunbar JC, Reinholt L, Henry RL, Mammen E. Platelet aggregation and
disaggregation in the streptozotocin induced diabetic rat: the effect of
sympathetic inhibition. Diabetes Res Clin Pract 1990;9:265–72.
[15] Nagata M, Messora M, Okamoto R, Campos N, Pola N, Esper L, et al. Influence
of the proportion of particulate autogenous bone graft/platelet-rich plasma
on bone healing in critical-size defects: an immunohistochemical analysis in
rat calvalia. Bone 2009;45:339–45.
[16] Iwai S, Kuyama K, Kuboyama N, Takiguchi S, Ogura N, Yamamoto H, et al.
Osteogenic potential of human dental follicle cells on rat calvaria. J Hard
Tissue Biol 2013;22:95–104.
[17] Takiguchi S, Kuboyama N, Kuyama K, Yamamoto H, Kondoh T. Experimental
study of bone formation ability with the periosteum on rat calvalia. J Hard
Tissue Biol 2009;18:149–60.
[18] Eda T, Takahashi K, Iwai S, Ogura N, Ito K, Tsukahara H, et al. Effects of plasma
rich in growth factors on bone formation in rat calvaria. J Hard Tissue Biol
2015;24:61–8.
[19] Suemitsu M, Utunomiya T, Yamamoto H. Histopathological image analysis
using the imageJ-Digitization of a component ratio-. Clin Med Pathol
2012;30:95–8.
[20] Zhao WJ, Duan F, Li ZT, Yang HJ, Huang Q, Wu KL. Evaluation of regional
bulbar redness using an image-based objective method. Int J Ophthalmol
2014;18:71–6.
[21] Kwapiszewska G, Chwalek K, Marsh LM, Wygrecka M, Whilhelm J, Best J, et al.
BDNF/TrkB signaling augments smooth muscle cell proliferation in
pulmonary hypertension. Am J Pathol 2012;181:2018–29.
[22] Shirakabe K, Terasawa K, Miyama K, Shibuya H, Nishida E. Regulation of the
activity of the transcription factor Runx2 by two homeobox proteins, Msx2
and Dlx5. Genes Cells 2001;6:851–6.
[23] Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, et al.
Stage-specific expression of Dlx-5 during osteoblast differentiation:
involvement in regulation of osteocalcin gene expression. Mol Endocrinol
1997;11:1681–94.
[24] Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The
novel zinc finger-containing transcription factor osterix is required for
osteoblast differentiation and bone formation. Cell 2002;108:17–29.
[25] Komori T. Regulation of skeletal development by the Runx family of
transcription factors. J Cell Biochem 2005;95:445–53.
[26] Takahashi K, Ogura N, Aonuma H, Ito K, Ishigami D, Kamino Y, et al. Bone
morphogenetic protein 6 stimulates mineralization in human dental follicle
cells without dexamethasone. Arch Oral Biol 2013;58:690–8.
[27] Tanaka S, Matsuzaka K, Sato D, Inoue T. Characteristics of newly formed bone
during guided bone regeneration: analysis of cbfa-1, osteocalcin, and VEGF
expression. J Oral Implantol 2007;33:321–6.
[28] Hawthorne AC, Xavier SP, Okamoto R, Salvador SL, Antunes AA, Salata LA.
Immunohistochemical, tomographic, and histological study on onlay bone
graft remodeling. Part III: allografts. Clin Oral Implants Res 2013;24:1164–72.
[29] Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, et al. Increased bone
formation in osteocalsin-deficient mice. Nature 1996;382:448–52.
[30] Hering S, Isken E, Knabbe C, Janott J, Jost C, Pommer A, et al. TGFbeta1 and
TGFbeta2 mRNA and protein expression in human bone samples. Exp Clin
Endocrinol Diabetes 2001;109:217–26.
[31] Vitsky A, Warie J, Pawliuk R, Bond A, Matthews D, Lacasse E, et al.
Homeostatic role of transforming growth factor- beta in the oral cavity and
esophagus of mice and its expression by mast cells in these tissues. Am J
Pathol 2009;174:2137–49.
[32] Letterio JJ, Roberts AB. Regulation of immune respnses by TGF- beta. Annu
Rev Immunol 1998;16:137–61.
[33] Bensamoun SF, Hawse JR, Subramaniam M, I.Iharreborde B, Bassillais A,
Benhamou CL, et al. TGFbeta inducible early gene-1 knockout mice display
defects in bone strength and microarchitecture. Bone 2006;39:1244–51.
[34] Andrew JG, Hoyland JA, Freemont AJ, Marsh DR. Platelet-derived growth
factor expression in normally healing human fractures. Bone 1995;16:455–60.
[35] Weiss S, Zimmermann G, Pufe T, Varoga D, Henle P. The systemic angiogenic
response during bone healing. Arch Orthop Trauma Surg 2009;129:989–97.
[36] Xian L, Wu X, Pang L, Lou M, Rosen CJ, Qiu T, et al. Matrix IGF-1 maintains
bone mass by activation of mTOR in mesenchymal stem cell. Nat Med
2012;18, 1095–01.
[37] Weiss S, Henle P, Bidlingmaier M, Moghaddam A, Kasten P, Zimmermann G.
Systemic response of the GH/IGF-I axis in timely versus delayed fracture
healing. Growth Horm IGF Res 2008;18:205–12.
[38] Flad HD, Brandt E. Platelet-derived chemokines: pathophysiology and
therapeutic aspects. Cell Mol Life Sci 2010;67:2363–86.
[39] Fréchette JP, Martineau I, Gagnon G. Platelet-rich plasmas: growth factor
content and roles in wound healing. J Dent Res 2005;84:434–9.
[40] Hartwig D, Härtel C, Henning H, Müller-Steinhardt M, Schlenke P, Klüter H.
Evidence for de novo synthesis of cytokines and chemokines in platelet
concentrates. Vox Sang 2002;82:182–90.