Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7

Contents lists available at ScienceDirect

Journal of Cranio-Maxillo-Facial Surgery

journal homepage: www.jcmfs.com

The influence of platelet-rich fibrin on angiogenesis in guided bone

regeneration using xenogenic bone substitutes: A study of rabbit
cranial defects
Jong-Suk Yoon, Sang-Hwa Lee, Hyun-Joong Yoon*
Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, Seoul, Republic of Korea

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

Article history: Purpose: The purpose of this study was to investigate the influence of platelet-rich fibrin (PRF) on
Paper received 10 July 2013 angiogenesis and osteogenesis in guided bone regeneration (GBR) using xenogenic bone in rabbit cranial
Accepted 7 January 2014 defects.
Materials and methods: In each rabbit, 2 circular bone defects, one on either side of the midline, were
Keywords: prepared using a reamer drill. Each of the experimental sites received bovine bone with PRF, and each of
PRF (platelet-rich fibrin)
the control sites received bovine bone alone. The animals were sacrificed at 1 week (n ¼ 4), 2 weeks
Vascular endothelial growth factor
(n ¼ 3) and 4 weeks (n ¼ 3). Biopsy samples were examined histomorphometrically by light microscopy,
Non-organic bovine bone
and expression of vascular endothelial growth factor (VEGF) was determined by immunohistochemical
staining.
Results: At all experimental time points, immunostaining intensity for VEGF was consistently higher in
the experimental group than in the control group. However, the differences between the control group
and the experimental group were not statistically significant in the histomorphometrical and immu-
nohistochemical examinations.
Conclusions: The results of this study suggest that PRF may increase the number of marrow cells.
However, PRF along with xenogenic bone substitutes does not show a significant effect on bony
regeneration. Further large-scale studies are needed to confirm our results.
Ó 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights
reserved.

1. Introduction has been given to the use of platelet concentrates in reconstructive

surgery (Deuel et al., 1991; Cochran and Wozney, 1999; Sahni et al.,
The presence of adequate bone volume and quality is one of the 2000; Marx, 2001; Lacoste et al., 2003; Rybarczyk et al., 2003;
essential factors for achieving osseointegration of a dental implant. Marx, 2004).
Bone augmentation using the guided bone regeneration (GBR) can Platelet-rich fibrin (PRF) was first developed by Choukroun et al.
be used in patients with an inadequate osseous width or height. for specific use in oral and maxillofacial surgery and is a second-
Over the last decade, considerable attention has focused on the generation platelet concentrate which is prepared from centri-
potential application of growth factors to enhance the wound fuged blood. This technique requires neither anticoagulants, nor
healing process. Growth factors involved in angiogenesis and bovine thrombin, nor any other gelling agents. The PRF protocol
osteogenesis are diverse. Some of these growth factors are released makes it possible to collect a fibrin clot charged with serum and
by platelets and include 3 isomeres of platelet-derived growth platelets (Choukroun et al., 2006a, 2006b; Dohan et al., 2006a,
factor (PDGFaa, PDGFbb and PDGFab), 2 of the numerous trans- 2006b; Dohan Ehrenfest et al., 2009). Dohan Ehrenfest et al.
forming growth factors (TGFb1 and TGFb2), vascular endothelial (2009) reported that PRF membrane releases high quantities of 3
growth factor (VEGF) and epithelial growth factor. Special attention main growth factors, such as TGF-b1, PDGF-ab and VEGF, over 7
days, of which TGF-b1 and VEGF were produced during the whole
experimental time. PDGF has been used clinically for treatment of
* Corresponding author. Department of Oral and Maxillofacial Surgery, Yeouido osseous defects, but it has not been used specifically to promote
St. Mary’s Hospital, Catholic University of Korea, #62 Yeouido-dong, Yeong-
angiogenesis. VEGF is a more potent angiogenic growth factor and
deungpo-gu, Seoul 150-713, Republic of Korea. Tel.: þ82 2 3779 1094; fax: þ82 2
769 1689. thus may serve as a good candidate for this study. Additionally,
E-mail address: [email protected] (H.-J. Yoon). VEGF has been implicated in having direct chemotactic and

1010-5182/$ e see front matter Ó 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcms.2014.01.034

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
2 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7

mitogenic effects on osteoblasts and osteogenic cells. Thus, it could

have direct and indirect effects on bone regeneration as part of GBR
procedures (Kaigler et al., 2013). Although some studies have re-
ported the healing effects of PRF on skin wounds (Choukroun et al.,
2006a, 2006b), considerable controversy exists regarding the effect
on GBR using xenogenic bone substitutes in the membraneous
bone wounds. Calvarial bone has been used in the present study
because of its embryological, morphological, and physiological
similarities with the maxillofacial region. It is evident that the
rabbit model chosen to evaluate bone repair in this study belongs to
a low-order phylogenetic species with a characteristically high
potential for osteogenesis. Such models are often used because they
are easy to anaesthetise, inexpensive to buy and maintain, and
require little space (Majzoub et al., 1999; Nishimura et al., 2004).
The purpose of this study was to investigate the influence of PRF
on the angiogenesis and osteogenesis in GBR using xenogenic bone
substitutes in rabbit cranial defects.
Fig. 1. Two circular bone defects were prepared, one on each side of sagittal suture.
2. Materials and methods

2.1. Animals

Ten adult male New Zealand white rabbits, weighing between

2.8 and 3.5 kg were used in this study and were kept in individual
metal cages at room temperature. All rabbits were checked be-
forehand for health by a single veterinarian. Each rabbit was given
an acclimatization period of 2 weeks prior to each surgery.
The study was approved by the Committee on the Use and Care
of Animals and the Institutional Review Board (IRB) of the Catholic
University of Korea (CUMC-2011-0057-01).

2.2. Surgical procedures Fig. 2. Each of the experimental group rabbits received non-organic bovine bone with
PRF (right), and each of the control group rabbits received non-organic bovine bone
alone (left).
General anaesthesia was induced by using intramuscular in-
jection of tiletamine/zolazepam (0.4 ml/kg; Zoletil, Verbac Korea,
Seoul, Republic of Korea) combined with xylazine HCL (0.15 ml/kg; alone (Fig. 2). The amount of Bio-ossÒ was measured with a syringe.
Rompun, Bayer in Korea, Seoul, Republic of Korea). Before the sur- The experimental or control sites were assigned on a random basis.
gical procedure, each animal received a single subcutaneous dose of Subsequently, 1 custom-made titanium (Ti > 99.5%) dome with an
penicillin G (0.1 ml/kg; Gentamycin, Kukje Pharm, Seoul, Republic inner diameter of 7.0 mm and an inner height of 3.5 mm were
of Korea), ketoprofen (0.03 ml/kg; Bukwang pharm, Seoul, Republic tightly fitted over each prepared site creating a complete peripheral
of Korea) and glycopyrrolate (0.2 ml/kg; Mobinul, Myungmoom seal between the dome margin and the bone surface. Each dome
Pharm, Seoul, Republic of Korea). In addition, 0.5 ml of 2% lidocaine was equipped with a 0.5-mm wide horizontal peripheral flange to
(Xylestesin-A, 3M ESPE AG, Seefeld, Germany) was injected locally ensure stability (Fig. 3). Both the periosteum and the skin were
at the periphery of the surgical site. PRF was prepared according to repositioned and primary closure was achieved with resorbable
the protocol (Dohan et al., 2006a; Sunitha and Munirathnam, suture material (Vicryl, Ethicon, Somerville, NJ, USA).
2008). A 3-ml blood sample was collected from each rabbit and
drawn into 10-ml test tubes without an anticoagulant. The blood
was centrifuged immediately using a tabletop centrifuge (406 G,
GYROGEN, Daejeon, Republic of Korea) for 10 min at 3000 rpm
(approximately 400 g). PRF clot was retrieved and mixed with
xenogenic bone substitutes (Bio-ossÒ, Geistlich-Pharma, Wolhusen,
Switzerland). PRF was obtained in the form of a membrane by
squeezing out the fluids in the fibrin clot. Immediately before sur-
gery, the scalp was carefully shaved and disinfected with a povi-
done-iodine topical antiseptic. The skin and subcutaneous tissues
were incised in the middle of the rabbit calvarial bone, extending
from the frontal to the occipital bone. The periosteum was carefully
incised and dissected bilaterally, exposing the cortical bone in the
region. Then 2 standardized circular defects e one on either side of
the midline e were prepared in the bone under constant irrigation
with a 0.9% saline solution, one on each side of the midline, using a
reamer drill with a diameter of 7.0 mm and a depth of 3.0 mm
mounted on a low-speed handpiece (Fig. 1). Each of the experi-
mental sites received PRF membrane with 0.15 ml of xenogenic
bone substitutes, and each of the control sites received Bio-ossÒ Fig. 3. Two custom-made titanium caps were tightly fitted to the prepared slits.

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7 3

Postoperatively, the animals received 3 days of penicillin G Table 2

administered subcutaneously at a dose of 0.3 ml/kg of body weight Results of histomorphometric analysis of newly formed bone (H & E).

and ketoprofen at dose of 0.03 ml/kg of body weight. The animals Control Experimental P value
were sacrificed by intramuscular administration of tiletamine/ Percentage of the height of newly formed bone (HB)
zolazepam 0.4 ml/kg combined with xylazine HCL 0.15 ml/kg and Week 1 (n ¼ 8) 3.75  1.26 3.00(1.83) 0.6532
intravenous administration of potassium chloride (20 ml; KCL- [4(2e5)] [3(1e5)]
40inj., Daihan Pharm, Seoul, Republic of Korea) at: 1 week Week 2 (n ¼ 6) 5.00  0.00 5.00  0.00 0.9999
[5(5e5)] [5(5e5)]
(n ¼ 4), 2 weeks (n ¼ 3) and 4 weeks (n ¼ 3).
Week 4 (n ¼ 6) 5.00  0.00 5.00  0.00 0.9999
[5(5e5)] [5(5e5)]
2.3. Histological preparations Percent area of newly formed bone (AB)
Week 1 (n ¼ 8) 6.50  3.11 5.00  3.74 0.6612
[6.5(3e10)] [4.5(1e10)]
The skull bones were retrieved en bloc with the titanium domes in Week 2 (n ¼ 6) 40.00  10.00 30.00  10.00 0.3687
situ and immediately fixed in 10% neutral buffered formalin. After [40(30e50)] [30(20e40)]
embedding in paraffin, sagittal 4-mm-thick sections were cut through Week 4 (n ¼ 6) 51.67  12.58 63.33  23.09 0.6428
[50(40e65)] [50(50e90)]
the central part of each titanium cap. The sections were stained with
Amount of marrow cell formation (MCF)
haematoxylin and eosin, then examined by light microscopy. Week 1(n ¼ 8)
Not detected 4 2
Less e 2(100.00) e
2.4. Immunostaining procedure
Similar e e
More e e
Microtome sections were evaluated by immunohistochemical Week 2 (n ¼ 6)
staining to detect VEGF using a polink-2 plus mouse kit (D58-15, Less 3(100.00) 3(100.00) e
Golden Bridge International, Mukillteo, WA, USA). Microtome sec- Similar e e
More e e
tions were de-paraffinized using serial xylene and ethanol baths.
Week 4 (n ¼ 6)
After rehydration, slides were heated in a microwave oven with Less 3(100.00) 3(100.00) e
0.01 M citrate buffer for 3 min and were then treated with 3% H2O2 Similar e e
in methanol for 15 min at room temperature. Slides were washed 3 More e e
times with TBST (Tris Buffer Saline Tween, Triology buffer, Cell- Data presented as n(%) and mean  sd[median(range)].
marque, USA) at pH 7.4 for 3 min and then incubated for 1 h with P value; difference between control and experimental group by Fisher’s exact test
primary antibodies (VEGF, ab28775, Abcam, UK) at dilution of and Wilcoxon rank sum test.

1:250 at room temperature. Slides were again washed 3 times with

formed bone relative to the area bounded by the titanium dome
TBST for 3 min and then incubated with an enhancer reagent (HK
and parent bone; the area bounded by the titanium dome and
518-50K, BioGenex, San Ramon, CA, USA) for 10 min at room
parent bone was designated as 70 and expressed as a percentage of
temperature. After that, slides were incubated with polymer-HRP
the total area of the newly formed bone (0e70). Third, the amount
(HK 519-50K, BioGenex) for 10 min. After the final washing with
of marrow cell formation in the grafted area relative to the amount
TBST, slides were treated with di-aminobenzidine chromogen
in the normal rabbit calvarial bone was evaluated on a 3-point scale
(DAKO, Glostrup, Denmark) to obtain a dark brown staining for
of 1e3. The amount of marrow cell formation for each specimen
immunoreaction. Sections were counterstained with Mayer’s hae-
was expressed as (1) less-than, (2) similar-to or (3) more-than the
matoxylin for 1 min, dehydrated in ethanol, cleared in xylene and
normal rabbit calvarial bone.
mounted in Canada balsam. All slides were evaluated by an inde-
Fourth, the expression of VEGF was evaluated as the percentage
pendent observer using a light microscope (Olympus BX 51,
of positively stained cells and the immunostaining intensity by
Olympus, Tokyo, Japan). The observer was blinded to the group and
using a 4-point scale of 0e3. After the immunostaining intensity of
relative age of the specimen.
the marrow cells of the normal rabbit calvarial bone was designated
as 3, the immunostaining intensity for each specimen was evalu-
2.5. Histomorphometric analysis ated by comparing it with the immunostaining intensity of the
marrow cells of the normal rabbit calvarial bone.
The histomorphometric data for the central section obtained The statistical significance of differences between the groups
from each specimen were recorded using a computerized image was evaluated by Fisher’s exact test and Wilcoxon rank sum test at
analysis system (Pannoramic MIDI, 3DHISTECH Ltd, Hungary). The P < 0.05.
newly formed bones within the experimental and control domes
were subjected to the following histomorphometric measure- 3. Results
ments. First, we determined the longest vertical height of the newly
generated tissue consisting of mineralized bone and marrow space 3.1. Histomorphometric evaluation
from the parent bone relative to the inner height of the titanium
dome; the inner height of the titanium dome was designated as 5 3.1.1. Percentage of the height of newly formed bone
and expressed as a percentage of the height of newly formed bone The percentage of height of the newly formed bone (HB) was
(0e5). Second, we calculated the percent of the area of the newly greater in the control group (3.75) than in the experimental group
(3.0) 1 week after surgery. The HB was 5.0 in both groups 2 and 4
Table 1
weeks after surgery. The difference was not statistically significant
Number of animals by week point.
between the control group and the experimental group (Tables 1
Control (N ¼ 10) Experimental (N ¼ 10) and 2; Figs. 4 and 5).
Week 1 4 (40.00) 4 (40.00)
Week 2 3 (30.00) 3 (30.00) 3.1.2. Percent area of newly formed bone
Week 4 3 (30.00) 3 (30.00) The percent area of newly formed bone (AB) was higher in the
Data presented as n (%). control group (6.5/40.0) than in the experimental group (5.0/30.0)

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
4 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7

Fig. 4. (A) Histologic findings of the control and (B) experimental group 1 week after surgery showing newly formed tissues (blue arrows) and Bio-Oss particles (yellow arrows)
beyond the external calvarial bone (H & E; 100).

1 and 2 weeks after surgery. However, the AB was greater in the 3.2.2. Percentage of positively stained cells
experimental group (63.33) than in the control group (51.67) 4 Significant differences in the percentage of positively stained
weeks after surgery. A linear increase in AB was seen in both the cells (PSC) were not seen between the experimental and control
experimental and control groups, whereas, no statistical difference groups over time (Fig. 10).
was noted between the control group and the experimental group
(Table 2; Fig. 6). 4. Discussion

3.1.3. Amount of marrow cell formation Some studies have found that the threshold of bridging for bone
Although the amount of marrow cell formation (MCF) was 0.5 in defects is about 1 mm in diameter without a membrane and 5 mm
the experimental group, no MCF was seen in the control group 1 diameter with a membrane or bone graft (Nishimura et al., 2004).
week after surgery. The MCF was 1.0 in both groups 2 and 4 weeks In our study, a circular defect was prepared in the bone with a
after surgery. No statistical difference was noted between the diameter of 7.0 mm because a custom-made titanium dome and
control group and the experimental group (Table 2; Figs. 7 and 8). xenogenic bone substitutes were used.
Sufficient space providing stiffness and occlusion of the
3.2. VEGF expression membrane must be created and maintained for an adequate
period of time during healing for an acceptable outcome of GBR
3.2.1. Immunostaining intensity (Nishimura et al., 2004). Our experimental model therefore used a
Immunostaining intensity was consistently higher in the custom-made titanium dome that allowed close adaptation and
experimental group (2.0/2.33/2.67) than in the control group (1.5/ stabilization of bone tissue, thereby inducing more complete bone
1.33/1.67) 1, 2 and 4 weeks after surgery. However, no statistical filling.
difference was noted between the control group and the experi- After preliminary studies on the use of platelet concentrates in
mental group. A linear increase in immunostaining intensity was maxillofacial surgery, this technique has been applied in numerous
seen in the experimental group (Table 3; Figs. 8 and 9).

Fig. 5. Results of histomorphometric analysis of the percentage of the height of newly Fig. 6. Results of histomorphometric analysis of the percent area of newly formed
formed bone (H & E). bone (H & E).

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7 5

Fig. 7. Results of histomorphometric analysis of marrow cell formation (H & E).

clinical situations. Choukroun’s PRF is considered a second- contact with the glass surface, so for successful preparation of PRF
generation platelet concentrate (Dohan Ehrenfest et al., 2009). speedy blood collection and immediate centrifugation, before the
This second-generation platelet concentrate eliminates the risks clotting cascade is initiated, is absolutely essential (Sunitha and
associated with the use of bovine thrombin. Because of the absence Munirathnam, 2008). If the time required to collect blood and
of an anticoagulant, blood begins to coagulate as soon as it comes in launch centrifugation is overly long, failure will occur.

Fig. 8. Marrow cell expression in VEGF immunostaining1 week after surgery. (A) Control site. No marrow cells are seen. (B) Experimental site. Marrow cells (yellow arrows) and
Osteoblasts (blue arrows) are seen around Bio-Oss particles. (C) Experimental site. VEGF expressions are seen in fat cells (yellow arrow) and endopoietic cells (green arrow).

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
6 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7

Table 3 angiogenesis and osteogenesis after animal long bone injury (Wang
Analysis of VEGF immunostaining. et al., 2001a, 2001b, 2011; Uchida et al., 2003). Steinbrech et al.
Control Experimental P value (2000) have pointed out that VEGF regulation leads to increased
Immunostaining intensity
angiogenesis in the hypoxic microenvironment of healing bone. In
Week 1 (n ¼ 8) our study, immunostaining intensity for VEGF was consistently
1 2(50.00) 1(25.00) 0.9999 higher in the experimental group than in the control group at all
2 2(50.00) 2(50.00) experimental time points. However, the differences between the
3 0(0.00) 1(25.00)
control group and the experimental group were not statistically
Week 2 (n ¼ 6)
1 2(66.67) 0(0.00) 0.4000 significant in the histomorphometrical and immunohistochemical
2 1(33.33) 2(66.67) examinations.
3 0(0.00) 1(33.33) The limitation of our study is that the amount of Bio-ossÒ was
Week 4 (n ¼ 6)
measured with a syringe. We put Bio-ossÒ into the syringe and
1 2(66.67) 0(0.00) 0.4000
2 1(33.33) 2(66.67)
tapped the syringe on the table gently to bring a surface to a level.
3 0(0.00) 1(33.33) However, this method can be inaccurate because of the porosity of
Percentage of positively stained cells the material.
Week 1(n ¼ 8) 100.00  0.00 97.50  5.00 0.4533
[100(100e100)] [100(90e100)]
Week 2 (n ¼ 6) 100.00  0.00 100.00  0.00 0.9999
[100(100e100)] [100(100e100)]
Week 4 (n ¼ 6) 100.00  0.00 100.00  0.00 0.9999
[100(100e100)] [100(100e100)]

Data presented as n(%).

P value; difference between control and experimental group by Fisher’s exact test
and Wilcoxon rank sum test.

Three biological phases are separated: (1) a coagulated red cell

layer at the bottom of the centrifugation tube, (2) a rigid and elastic
PRF gel as intermediate layer that is squeezed to obtain a fibrin
membrane and used for clinical applications, and (3) a liquid su-
pernatant serum that is discarded (Su et al., 2009). Platelets isolated
from peripheral blood are an autologous source of growth factors.
When platelets in a concentrated form are added to graft materials,
a more predictable outcome is derived (Sunitha and Munirathnam,
2008). Su et al. (2009) have pointed out that the PRF membrane
should be used immediately after formation to maximize release of
growth factors to the surgical site and the remaining fluid can be
recovered as an additional source of growth factors for grafting.
Choukroun et al. (2006b) have documented that PRF does not
appear to enhance cellular proliferation in the long term but may
play an important role in the revascularization of the graft by
supporting angiogenesis. They also reported that sinus floor
augmentation with FDBA and PRF reduces healing time prior to
implant placement. Lee et al. (2007) have demonstrated, in an
animal study, that clinical outcomes are better using autogenous
bone mixed with PRF than using autogenous bone alone. Park et al.
(2011) evaluated the effect of PRP on early bone regeneration of
rabbit cranial defects when used in combination with beta-trical-
cium phosphate and reported that PRF along with beta-tricalcium
phosphate shows a significant effect on bone regeneration.
Although numerous studies have been conducted to evaluate the
effects of PRF on the bone healing process (Suttaapreyasri and
Leepong, 2013; Zhang et al., 2012), there have been few studies
on the influence of PRF on angiogenesis and osteogenesis in GBR
using xenogenic bone substitutes in rabbit cranial defects. In our
study, the number of marrow cells was higher in the experimental
group than in the control group 1 week after surgery. This indicates
that the number of marrow cells reach that of normal rabbit cal-
varial bone more rapidly in the experimental group than in the
control group.
Angiogenesis consists of the formation of new blood vessels
inside the wound. It has been clearly demonstrated that the fibrin
matrix leads directly to angiogenesis (Choukroun et al., 2006b).
Angiogenesis underlies the success of guided bone regeneration
(GBR) procedures. Previous studies have indicated that VEGF plays
a crucial role in the healing process through modulation of Fig. 9. Immunostaining intensity of VEGF.

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034
 J.-S. Yoon et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7 7

Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al: Platelet-rich
fibrin (PRF): a second-generation platelet concentrate. Part I: technological
concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:
e37e44, 2006a
Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al: Platelet-rich
fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related
biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:
e45e50, 2006b
Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G: Slow release of
growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin
(PRF): a gold standard to achieve for all surgical platelet concentrates tech-
nologies. Growth Factors 27: 63e69, 2009
Kaigler D, Silva EA, Mooney DJ: Guided bone regeneration using injectable vascular
endothelial growth factor delivery gel. J Periodontol 84: 230e238, 2013
Lacoste E, Martineau I, Gagnon G: Platelet concentrates: effects of calcium and
thrombin on endothelial cell proliferation and growth factor release.
J Periodontol 74: 1498e1507, 2003
Lee HJ, Choi BH, Jung JH, Zhu SJ, Lee SH, Huh JY, et al: Maxillary sinus floor
augmentation using autogenous bone grafts and platelet-enriched fibrin glue
with simultaneous implant placement. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 103: 329e333, 2007
Majzoub Z, Berengo M, Giardino R, Aldini NN, Cordioli G: Role of intramarrow
penetration in osseous repair: a pilot study in the rabbit calvaria. J Periodontol
70: 1501e1510, 1999
Marx RE: Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant
Dent 10: 225e228, 2001
Marx RE: Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg
Fig. 10. Percentage of positively stained cells of VEGF immunostaining. 62: 489e496, 2004
Nishimura I, Shimizu Y, Ooya K: Effects of cortical bone perforation on experimental
guided bone regeneration. Clin Oral Implants Res 15: 293e300, 2004
Park JK, Joo HJ, Lee ES, Jang HS, Lim JS, Kwon JJ: The effect of PRF and PRP for new
5. Conclusions bone formation of beta-TCP in skull of white rabbit. J Korean Assoc Maxillofac
Plast Reconstr Surg 33: 19e25, 2011
The results of this study suggest that PRF may increase the Rybarczyk BJ, Lawrence SO, Simpson-Haidaris PJ: Matrix-fibrinogen enhances
wound closure by increasing both cell proliferation and migration. Blood 102:
number of marrow cells, but PRF along with xenogenic bone sub- 4035e4043, 2003
stitutes does not show a significant effect on bony regeneration. Sahni A, Baker CA, Sporn LA, Francis CW: Fibrinogen and fibrin protect fibroblast
Further large-scale studies are needed to confirm our results. growth factor-2 from proteolytic degradation. Thromb Haemost 83: 736e741,
2000
Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Gittes GK, et al:
Funding source VEGF expression in an osteoblast-like cell line is regulated by a hypoxia
None. response mechanism. Am J Physiol Cell Physiol 278: C853eC860, 2000
Su CY, Kuo YP, Tseng YH, Su CH, Burnouf T: In vitro release of growth factors from
platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.
Conflict of interest statement Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108: 56e61, 2009
The authors declare that there is no conflict of interest. Sunitha RV, Munirathnam NE: Platelet-rich fibrin: evolution of a second-generation
platelet concentrate Indian. J Dent Res 19: 42e46, 2008
Suttaapreyasri S, Leepong N: Influence of platelet-rich fibrin on alveolar ridge
Acknowledgements preservation. J Craniofac Surg 24: 1088e1094, 2013
Uchida S, Sakai A, Kudo H, Otomo H, Watanuki M, Tanaka M, et al: Vascular
None. endothelial growth factor is expressed along with its receptors during the
healing process of bone and bone marrow after drill-hole injury in rats. Bone
32: 491e501, 2003
References Wang CJ, Chen HS, Chen CE, Yang KD: Treatment of nonunions of long bone frac-
tures with shock waves. Clin Orthop Relat Res: 95e101, 2001a
Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al: Platelet- Wang CJ, Huang HY, Chen HH, Pai CH, Yang KD: Effect of shock wave therapy on
rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical ef- acute fractures of the tibia: a study in a dog model. Clin Orthop Relat Res: 112e
fects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101: 118, 2001b
e56e60, 2006a Wang CJ, Huang KE, Sun YC, Yang YJ, Ko JY, Weng LH, et al: VEGF modulates
Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al: Platelet- angiogenesis and osteogenesis in shockwave-promoted fracture healing in
rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic rabbits. J Surg Res 171: 114e119, 2011
evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Zhang Y, Tangl S, Huber CD, Lin Y, Qiu L, Rausch-Fan X: Effects of Choukroun’s
Oral Med Oral Pathol Oral Radiol Endod 101: 299e303, 2006b platelet-rich fibrin on bone regeneration in combination with deproteinized
Cochran DL, Wozney JM: Biological mediators for periodontal regeneration. Perio- bovine bone mineral in maxillary sinus augmentation: a histological and his-
dontol 2000 19: 40e58, 1999 tomorphometric study. J Craniomaxillofac Surg 40: 321e328, 2012
Deuel TF, Kawahara RS, Mustoe TA, Pierce AF: Growth factors and wound healing:
platelet-derived growth factor as a model cytokine. Annu Rev Med 42: 567e
584, 1991

Please cite this article in press as: Yoon J-S, et al., The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using
xenogenic bone substitutes: A study of rabbit cranial defects, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/
j.jcms.2014.01.034