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 xxx (2014) 1e7

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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: Paper received 10 July 2013 Accepted 7 January 2014

Purpose: The purpose of this study was to investigate the influence of platelet-rich fibrin (PRF) on angiogenesis and osteogenesis in guided bone regeneration (GBR) using xenogenic bone in rabbit cranial defects. Materials and methods: In each rabbit, 2 circular bone defects, one on either side of the midline, were prepared using a reamer drill. Each of the experimental sites received bovine bone with PRF, and each of the control sites received bovine bone alone. The animals were sacrificed at 1 week (n ¼ 4), 2 weeks (n ¼ 3) and 4 weeks (n ¼ 3). Biopsy samples were examined histomorphometrically by light microscopy, 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 immunohistochemical 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.

Keywords: PRF (platelet-rich fibrin) Vascular endothelial growth factor Non-organic bovine bone

1. Introduction The presence of adequate bone volume and quality is one of the essential factors for achieving osseointegration of a dental implant. Bone augmentation using the guided bone regeneration (GBR) can be used in patients with an inadequate osseous width or height. Over the last decade, considerable attention has focused on the potential application of growth factors to enhance the wound healing process. Growth factors involved in angiogenesis and osteogenesis are diverse. Some of these growth factors are released by platelets and include 3 isomeres of platelet-derived growth factor (PDGFaa, PDGFbb and PDGFab), 2 of the numerous transforming growth factors (TGFb1 and TGFb2), vascular endothelial growth factor (VEGF) and epithelial growth factor. Special attention

* Corresponding author. Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, #62 Yeouido-dong, Yeongdeungpo-gu, Seoul 150-713, Republic of Korea. Tel.: þ82 2 3779 1094; fax: þ82 2 769 1689. E-mail address: [email protected] (H.-J. Yoon).

has been given to the use of platelet concentrates in reconstructive surgery (Deuel et al., 1991; Cochran and Wozney, 1999; Sahni et al., 2000; Marx, 2001; Lacoste et al., 2003; Rybarczyk et al., 2003; Marx, 2004). Platelet-rich fibrin (PRF) was first developed by Choukroun et al. for specific use in oral and maxillofacial surgery and is a secondgeneration platelet concentrate which is prepared from centrifuged blood. This technique requires neither anticoagulants, nor bovine thrombin, nor any other gelling agents. The PRF protocol makes it possible to collect a fibrin clot charged with serum and platelets (Choukroun et al., 2006a, 2006b; Dohan et al., 2006a, 2006b; Dohan Ehrenfest et al., 2009). Dohan Ehrenfest et al. (2009) reported that PRF membrane releases high quantities of 3 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 osseous defects, but it has not been used specifically to promote angiogenesis. VEGF is a more potent angiogenic growth factor and thus may serve as a good candidate for this study. Additionally, 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

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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 reported 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 beforehand 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 General anaesthesia was induced by using intramuscular injection of tiletamine/zolazepam (0.4 ml/kg; Zoletil, Verbac Korea, Seoul, Republic of Korea) combined with xylazine HCL (0.15 ml/kg; Rompun, Bayer in Korea, Seoul, Republic of Korea). Before the surgical procedure, each animal received a single subcutaneous dose of penicillin G (0.1 ml/kg; Gentamycin, Kukje Pharm, Seoul, Republic of Korea), ketoprofen (0.03 ml/kg; Bukwang pharm, Seoul, Republic of Korea) and glycopyrrolate (0.2 ml/kg; Mobinul, Myungmoom Pharm, Seoul, Republic of Korea). In addition, 0.5 ml of 2% lidocaine (Xylestesin-A, 3M ESPE AG, Seefeld, Germany) was injected locally at the periphery of the surgical site. PRF was prepared according to the protocol (Dohan et al., 2006a; Sunitha and Munirathnam, 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 surgery, the scalp was carefully shaved and disinfected with a povidone-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 experimental sites received PRF membrane with 0.15 ml of xenogenic bone substitutes, and each of the control sites received Bio-ossÒ

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).

alone (Fig. 2). The amount of Bio-ossÒ was measured with a syringe. The experimental or control sites were assigned on a random basis. Subsequently, 1 custom-made titanium (Ti > 99.5%) dome with an inner diameter of 7.0 mm and an inner height of 3.5 mm were tightly fitted over each prepared site creating a complete peripheral seal between the dome margin and the bone surface. Each dome was equipped with a 0.5-mm wide horizontal peripheral flange to ensure stability (Fig. 3). Both the periosteum and the skin were repositioned and primary closure was achieved with resorbable suture material (Vicryl, Ethicon, Somerville, NJ, USA).

Fig. 3. Two custom-made titanium caps were tightly fitted to the prepared slits.



Postoperatively, the animals received 3 days of penicillin G administered subcutaneously at a dose of 0.3 ml/kg of body weight and ketoprofen at dose of 0.03 ml/kg of body weight. The animals were sacrificed by intramuscular administration of tiletamine/ zolazepam 0.4 ml/kg combined with xylazine HCL 0.15 ml/kg and intravenous administration of potassium chloride (20 ml; KCL40inj., Daihan Pharm, Seoul, Republic of Korea) at: 1 week (n ¼ 4), 2 weeks (n ¼ 3) and 4 weeks (n ¼ 3). 2.3. Histological preparations The skull bones were retrieved en bloc with the titanium domes in situ and immediately fixed in 10% neutral buffered formalin. After embedding in paraffin, sagittal 4-mm-thick sections were cut through the central part of each titanium cap. The sections were stained with haematoxylin and eosin, then examined by light microscopy. 2.4. Immunostaining procedure Microtome sections were evaluated by immunohistochemical staining to detect VEGF using a polink-2 plus mouse kit (D58-15, Golden Bridge International, Mukillteo, WA, USA). Microtome sections were de-paraffinized using serial xylene and ethanol baths. After rehydration, slides were heated in a microwave oven with 0.01 M citrate buffer for 3 min and were then treated with 3% H2O2 in methanol for 15 min at room temperature. Slides were washed 3 times with TBST (Tris Buffer Saline Tween, Triology buffer, Cellmarque, USA) at pH 7.4 for 3 min and then incubated for 1 h with primary antibodies (VEGF, ab28775, Abcam, UK) at dilution of 1:250 at room temperature. Slides were again washed 3 times with TBST for 3 min and then incubated with an enhancer reagent (HK 518-50K, BioGenex, San Ramon, CA, USA) for 10 min at room temperature. After that, slides were incubated with polymer-HRP (HK 519-50K, BioGenex) for 10 min. After the final washing with TBST, slides were treated with di-aminobenzidine chromogen (DAKO, Glostrup, Denmark) to obtain a dark brown staining for immunoreaction. Sections were counterstained with Mayer’s haematoxylin for 1 min, dehydrated in ethanol, cleared in xylene and mounted in Canada balsam. All slides were evaluated by an independent observer using a light microscope (Olympus BX 51, Olympus, Tokyo, Japan). The observer was blinded to the group and relative age of the specimen. 2.5. Histomorphometric analysis The histomorphometric data for the central section obtained from each specimen were recorded using a computerized image analysis system (Pannoramic MIDI, 3DHISTECH Ltd, Hungary). The newly formed bones within the experimental and control domes were subjected to the following histomorphometric measurements. First, we determined the longest vertical height of the newly generated tissue consisting of mineralized bone and marrow space from the parent bone relative to the inner height of the titanium dome; the inner height of the titanium dome was designated as 5 and expressed as a percentage of the height of newly formed bone (0e5). Second, we calculated the percent of the area of the newly Table 1 Number of animals by week point.

Week 1 Week 2 Week 4 Data presented as n (%).

Control (N ¼ 10)

Experimental (N ¼ 10)

4 (40.00) 3 (30.00) 3 (30.00)

4 (40.00) 3 (30.00) 3 (30.00)

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Table 2 Results of histomorphometric analysis of newly formed bone (H & E). Control

Experimental

Percentage of the height of newly formed bone (HB) Week 1 (n ¼ 8) 3.75 1.26 3.00(1.83) [4(2e5)] [3(1e5)] Week 2 (n ¼ 6) 5.00 0.00 5.00 0.00 [5(5e5)] [5(5e5)] Week 4 (n ¼ 6) 5.00 0.00 5.00 0.00 [5(5e5)] [5(5e5)] Percent area of newly formed bone (AB) Week 1 (n ¼ 8) 6.50 3.11 5.00 3.74 [6.5(3e10)] [4.5(1e10)] Week 2 (n ¼ 6) 40.00 10.00 30.00 10.00 [40(30e50)] [30(20e40)] Week 4 (n ¼ 6) 51.67 12.58 63.33 23.09 [50(40e65)] [50(50e90)] Amount of marrow cell formation (MCF) Week 1(n ¼ 8) Not detected 4 2 Less e 2(100.00) Similar e e More e e Week 2 (n ¼ 6) Less 3(100.00) 3(100.00) Similar e e More e e Week 4 (n ¼ 6) Less 3(100.00) 3(100.00) Similar e e More e e

P value 0.6532 0.9999 0.9999

0.6612 0.3687 0.6428

e

e

e

Data presented as n(%) and mean sd[median(range)]. P value; difference between control and experimental group by Fisher’s exact test and Wilcoxon rank sum test.

formed bone relative to the area bounded by the titanium dome and parent bone; the area bounded by the titanium dome and parent bone was designated as 70 and expressed as a percentage of the total area of the newly formed bone (0e70). Third, the amount of marrow cell formation in the grafted area relative to the amount in the normal rabbit calvarial bone was evaluated on a 3-point scale of 1e3. The amount of marrow cell formation for each specimen was expressed as (1) less-than, (2) similar-to or (3) more-than the normal rabbit calvarial bone. Fourth, the expression of VEGF was evaluated as the percentage of positively stained cells and the immunostaining intensity by using a 4-point scale of 0e3. After the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone was designated as 3, the immunostaining intensity for each specimen was evaluated by comparing it with the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone. The statistical significance of differences between the groups was evaluated by Fisher’s exact test and Wilcoxon rank sum test at P < 0.05. 3. Results 3.1. Histomorphometric evaluation 3.1.1. Percentage of the height of newly formed bone The percentage of height of the newly formed bone (HB) was 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 weeks after surgery. The difference was not statistically significant between the control group and the experimental group (Tables 1 and 2; Figs. 4 and 5). 3.1.2. Percent area of newly formed bone The percent area of newly formed bone (AB) was higher in the control group (6.5/40.0) than in the experimental group (5.0/30.0)


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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 experimental group (63.33) than in the control group (51.67) 4 weeks after surgery. A linear increase in AB was seen in both the experimental and control groups, whereas, no statistical difference was noted between the control group and the experimental group (Table 2; Fig. 6). 3.1.3. Amount of marrow cell formation Although the amount of marrow cell formation (MCF) was 0.5 in the experimental group, no MCF was seen in the control group 1 week after surgery. The MCF was 1.0 in both groups 2 and 4 weeks after surgery. No statistical difference was noted between the control group and the experimental group (Table 2; Figs. 7 and 8). 3.2. VEGF expression 3.2.1. Immunostaining intensity Immunostaining intensity was consistently higher in the experimental group (2.0/2.33/2.67) than in the control group (1.5/ 1.33/1.67) 1, 2 and 4 weeks after surgery. However, no statistical difference was noted between the control group and the experimental group. A linear increase in immunostaining intensity was 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 formed bone (H & E).

3.2.2. Percentage of positively stained cells Significant differences in the percentage of positively stained cells (PSC) were not seen between the experimental and control groups over time (Fig. 10). 4. Discussion Some studies have found that the threshold of bridging for bone defects is about 1 mm in diameter without a membrane and 5 mm diameter with a membrane or bone graft (Nishimura et al., 2004). In our study, a circular defect was prepared in the bone with a diameter of 7.0 mm because a custom-made titanium dome and xenogenic bone substitutes were used. Sufficient space providing stiffness and occlusion of the membrane must be created and maintained for an adequate period of time during healing for an acceptable outcome of GBR (Nishimura et al., 2004). Our experimental model therefore used a custom-made titanium dome that allowed close adaptation and stabilization of bone tissue, thereby inducing more complete bone filling. After preliminary studies on the use of platelet concentrates in maxillofacial surgery, this technique has been applied in numerous

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



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

clinical situations. Choukroun’s PRF is considered a secondgeneration platelet concentrate (Dohan Ehrenfest et al., 2009). This second-generation platelet concentrate eliminates the risks associated with the use of bovine thrombin. Because of the absence of an anticoagulant, blood begins to coagulate as soon as it comes in

contact with the glass surface, so for successful preparation of PRF speedy blood collection and immediate centrifugation, before the clotting cascade is initiated, is absolutely essential (Sunitha and Munirathnam, 2008). If the time required to collect blood and 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).


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Table 3 Analysis of VEGF immunostaining. Control Immunostaining intensity Week 1 (n ¼ 8) 1 2(50.00) 2 2(50.00) 3 0(0.00) Week 2 (n ¼ 6) 1 2(66.67) 2 1(33.33) 3 0(0.00) Week 4 (n ¼ 6) 1 2(66.67) 2 1(33.33) 3 0(0.00) Percentage of positively stained cells Week 1(n ¼ 8) 100.00 0.00 [100(100e100)] Week 2 (n ¼ 6) 100.00 0.00 [100(100e100)] Week 4 (n ¼ 6) 100.00 0.00 [100(100e100)]

Experimental

P value

1(25.00) 2(50.00) 1(25.00)

0.9999

0(0.00) 2(66.67) 1(33.33)

0.4000

0(0.00) 2(66.67) 1(33.33)

0.4000

97.50 5.00 [100(90e100)] 100.00 0.00 [100(100e100)] 100.00 0.00 [100(100e100)]

0.4533

angiogenesis and osteogenesis after animal long bone injury (Wang et al., 2001a, 2001b, 2011; Uchida et al., 2003). Steinbrech et al. (2000) have pointed out that VEGF regulation leads to increased angiogenesis in the hypoxic microenvironment of healing bone. In our study, immunostaining intensity for VEGF was consistently higher in the experimental group than in the control group at all experimental time points. However, the differences between the control group and the experimental group were not statistically significant in the histomorphometrical and immunohistochemical examinations. The limitation of our study is that the amount of Bio-ossÒ was measured with a syringe. We put Bio-ossÒ into the syringe and tapped the syringe on the table gently to bring a surface to a level. However, this method can be inaccurate because of the porosity of the material.

0.9999 0.9999

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 supernatant 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-tricalcium 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 calvarial 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.



Fig. 10. Percentage of positively stained cells of VEGF immunostaining.

5. Conclusions The results of this study suggest that PRF may increase the number of marrow cells, but 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. Funding source None. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgements None. References Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al: Plateletrich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101: e56e60, 2006a Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al: Plateletrich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101: 299e303, 2006b Cochran DL, Wozney JM: Biological mediators for periodontal regeneration. Periodontol 2000 19: 40e58, 1999 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

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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 technologies. 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 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 bone formation of beta-TCP in skull of white rabbit. J Korean Assoc Maxillofac Plast Reconstr Surg 33: 19e25, 2011 Rybarczyk BJ, Lawrence SO, Simpson-Haidaris PJ: Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration. Blood 102: 4035e4043, 2003 Sahni A, Baker CA, Sporn LA, Francis CW: Fibrinogen and fibrin protect fibroblast 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: VEGF expression in an osteoblast-like cell line is regulated by a hypoxia 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. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108: 56e61, 2009 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 preservation. J Craniofac Surg 24: 1088e1094, 2013 Uchida S, Sakai A, Kudo H, Otomo H, Watanuki M, Tanaka M, et al: Vascular 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 Wang CJ, Chen HS, Chen CE, Yang KD: Treatment of nonunions of long bone fractures with shock waves. Clin Orthop Relat Res: 95e101, 2001a Wang CJ, Huang HY, Chen HH, Pai CH, Yang KD: Effect of shock wave therapy on acute fractures of the tibia: a study in a dog model. Clin Orthop Relat Res: 112e 118, 2001b Wang CJ, Huang KE, Sun YC, Yang YJ, Ko JY, Weng LH, et al: VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res 171: 114e119, 2011 Zhang Y, Tangl S, Huber CD, Lin Y, Qiu L, Rausch-Fan X: Effects of Choukroun’s platelet-rich fibrin on bone regeneration in combination with deproteinized bovine bone mineral in maxillary sinus augmentation: a histological and histomorphometric study. J Craniomaxillofac Surg 40: 321e328, 2012


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