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Reduction of bone resorption by the application of platelet-rich plasma (PRP) in bone grafting of the alveolar cleft
Journal of Cranio-Maxillo-Facial Surgery 39 (2011) 278e283
Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Reduction of bone resorption by the application of platelet-rich plasma (PRP) in bone grafting of the alveolar cleft Eriko Marukawa*, Hidekazu Oshina, Gaichi Iino, Keiichi Morita, Ken Omura Oral and Maxillofacial Surgery, Department of Oral Restitution, Division of Oral Health Sciences, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 9 December 2009 Accepted 23 April 2010
Objective: We evaluated the effectiveness of platelet-rich plasma (PRP) on the regeneration of autogenous cancellous bone and marrow grafted in the alveolar cleft. Design: Twenty patients with alveolar clefts were examined; 6 were the control group and received cancellous bone and marrow grafts without PRP, while the remaining 14 comprised the PRP group and received grafts with PRP. Prior to surgery, 50 ml of blood was withdrawn and 5 ml of PRP gel produced through centrifugal separation. The bone graft mixed with PRP was then packed into the alveolar cleft. Postoperative bone density was assessed as the aluminium-equivalent value on occlusal X-ray films in a qualitative analysis. Quantitative evaluation of regenerated bone was made with computed tomography and panoramic radiographs at 1 month, 6 months and 1 year after surgery. Results: Satisfactory bone bridging formation was observed in all patients without any complications. The bone density of the PRP group was lower than that of the control group at 1 week, but the same after 1 month. The added PRP reduced the resorption of regenerated bone postoperatively. Conclusion: Autogenous cancellous bone grafting with PRP, which significantly reduces postoperative bone resorption, is a reliable technique for alveolar bone grafting of cleft patients. Ó 2010 European Association for Cranio-Maxillo-Facial Surgery.
Keywords: Platelet-rich plasma (PRP) Alveolar bone grafting Alveolar cleft
1. Introduction In cleft lip and palate patients, bone grafting of the alveolar cleft is necessary for rehabilitation of oral function (Boyne and Sands, 1972; Witsenburg, 1985; Schendel, 1994; Keese and Schmelzle, 1995; Long et al., 1995). Among the available graft materials, autogenous bone is currently the preferred material. In some cleft patients it is difficult to augment the bone defect adequately because of the width of the gap between the maxillary segments, or as a result of bone resorption (Boyne and Sands,1972; Long et al.,1995; Opitz et al.,1999). A method for accelerating the speed of bone formation and reducing bone resorption in alveolar cleft bone grafting has been sought for some time. In 1998, platelet-rich plasma (PRP) was reported by Marx et al. (1998) to promote new bone formation in mandibular continuity defects and to cause faster maturation of autologous bone grafts. PRP contains a high concentration of platelets and is an autologous source of PDGF (platelet-derived growth factor), TGF-b, (transforming
* Corresponding author. Tel.: þ81 3 5803 5506; fax: þ81 3 5803 0199. E-mail address: [email protected] (E. Marukawa).
growth factor-b) and VEGF (vascular endothelial growth factor). Many reports (Marx et al., 1998; Aghaloo et al., 2002; Wiltfang et al., 2003; Aghaloo et al., 2004; Merkx et al., 2004; Schlegel et al., 2004; Graziani et al., 2005; Hokugo et al., 2005) have confirmed the effectiveness of PRP in enhancing bone regeneration in autologous bone grafts and other bone substitutes, although others have shown no benefit of PRP on bone formation (Choi et al., 2004; Wiltfang et al., 2004; Aghaloo et al., 2005; Pryor et al., 2005a,b; Raghoebar et al., 2005; Klongnoi et al., 2006a,b; Plachokova et al., 2006; Sarkar et al., 2006; Thorwarth et al., 2006). In this clinical study, we used autogenous cancellous bone with PRP in alveolar bone grafting of cleft patients in order to evaluate the effectiveness of PRP.
2. Materials and methods 2.1. Patients 20 non-syndromic patients with unilateral clefts treated with alveolar bone grafting during the period between March 2002 and March 2004 were included in this study. The patients were randomly assigned to two groups: the PRP group, who received
1010-5182/$ e see front matter Ó 2010 European Association for Cranio-Maxillo-Facial Surgery. doi:10.1016/j.jcms.2010.04.017
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autogenous cancellous bone and marrow grafts with PRP (14 patients; 7 males and 7 females, 11 unilateral cleft lip and palate and 3 cleft lip and alveolus); and the control group, who received grafts without PRP (6 patients; 2 males and 4 females, 1 unilateral cleft lip and palate and 5 cleft lip and alveolus). At the time of surgery the mean age of the patients was 20 4.7 years with a range 10e29 years. All procedures and materials were approved by the local Ethics Committee. Informed consent was obtained from all patients. 2.2. Preparation of PRP Prior to surgery 50 ml of blood was withdrawn from each patient with 5 ml of citrate phosphate dextrose (CPD: TERUMO, Japan) for anticoagulation. PRP gel was produced through centrifugal separation of whole blood. After the first centrifugation (800 g, 5 min), the blood was separated into plasma and red blood cells. The red blood cells were removed, and after a further centrifugation (1500 g, 5 min) of the remaining plasma, the bottom layer, which was rich in platelets and constituted approximately 10% of the total withdrawn blood volume, was collected for use as PRP. At the time of grafting, the solution was mixed with 2% calcium chloride at a volume ratio of 7 to 1 to start gelation and promote the release of growth factors, such as TGF-b, PDGFs, VEGF, and insulin-like growth factors (IGFs), from the platelets (Slater et al., 1995; Rendu and Brohard-Bohn, 2001; Eppley et al., 2004; Frechette et al., 2005). The TGF-b1, PDGF-AB and VEGF concentrations of each PRP preparation and whole blood were assayed using an ELISA (enzyme-linked immunosorbent assay) kit (Quantikaine, R&D Systems Inc., Minneapolis, MN, USA). 2.3. Surgical procedure Alveolar bone grafting was performed under general endotracheal anaesthesia. Incisions were made and gingival mucoperiosteal flaps were elevated in the standard fashion for alveolar bone grafting. Autogenous cancellous bone and marrow from the anterior iliac crest was used for each graft. The PRP gel was mixed with the bone graft to obtain a gel-like consistency (Fig. 1) and this mixture was then packed into the alveolar cleft and closed with gingival mucoperiosteal flaps. 2.4. Clinical and radiological evaluation Clinical and radiological follow-up examinations were carried out for 1 year. The quantity and quality of bone at the grafted sites were radiographically evaluated with the use of panoramic and occlusal radiographs and computed tomography (CT). Axial CT scans were obtained at a slice thickness of 1 mm. The level and window of each slice were optimally set to allow precise delineation between bone and soft tissue. Intra-oral radiographs of the grafted sites were taken to document progressive bone formation every month after surgery. We assessed the bone density of the grafted sites on occlusal X-ray films in a qualitative analysis. The measurement procedure consisted of image acquisition, correction for radiographic variation using an aluminium wedge (Fig. 2a) and transformation of the grey values into aluminium-equivalent values to produce the relative density of the bone. All relative density was subtracted from the pre-operative aluminium-equivalent values to correct for variables such as soft tissue density that can be confounding factors. Quantitative evaluation of the regenerated bone was made using the CT scans (Fig. 2b) and panoramic radiographs (Fig. 2c). The width of the alveolar crest was measured using a CT scan at 1 month, 6 months and 1 year after surgery. The width of grafted
Fig. 1. The application of PRP to autogenous bone particles. The cancellous marrow bone particles were mixed with the PRP for gel formation and activation of the PRP. They can be handled easily.
sites at 1 month was set as the baseline value, and the ratio of bone loss at 6 months and 1 year after surgery calculated. Using panoramic radiographs, the height of the alveolar crest was measured at 1 week, 1 month, 6 months and 1 year after surgery. The height of grafted sites at 1 week was set as the baseline value and the ratio of bone loss at 1 month, 6 months and 1 year after surgery calculated. The data for all the patients was obtained and analysed by two researchers who were blinded as to which treatment the patients had received. The average of the two sets of measurements for each patient was used for comparison of the two groups. The ManneWhitney U test revealed a statistical significant difference (P < 0.05) between the groups of PRP patients and controls.
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Fig. 2. Evaluation of the quantity and quality in grafted sites. (a) An aluminium step wedge was placed during all occlusal radiographic exposures. The thickness of the alveolar bone at each pixel was calculated as an equivalent aluminium thickness. (b) The width of the alveolar crest was measured on axial CT images. The mean bone loss (%) at 6 months and 1 year, compared to 1 month, was evaluated. (c) The height of the alveolar crest was measured on panoramic radiographs. The mean bone loss (%) at 1 month, 6 months and 1 year, compared to 1 week, was evaluated.
Fig. 3. (a) The concentrations of platelets in serum and PRP. (b) The concentrations of TGF-b1 in serum and PRP. (c) The concentrations of VEGF in serum and PRP. (d) The concentrations of PDGF-AB in serum and PRP. Platelet counts in PRP were about 3-fold greater, and growth factors were 2e3-fold greater, than in serum.
3. Results 3.1. Clinical findings The mean concentrations of platelets, TGF-b1, VEGF and PDGFAB were all higher in the PRP (Fig. 3aed), with platelets being about 3-fold higher, and platelet-released growth factors about 2e3-fold higher, than in serum. Of the 20 patients, 17 (control group, 4; PRP group, 13) had an uneventful course postoperatively. In the 3 remaining patients
(control group, 2; PRP group, 1), wound dehiscence developed with minor bone exposure. However, these exposures closed during the follow-up period. No other complications were observed. Bone formation was satisfactory in all patients. 3.2. Radiological evaluation The mean relative density of bone grafts aluminium-equivalent values (mmAl equivalent) and mean total bone loss in the regenerated bone are shown in Figs. 4e6. At 1 week after surgery, the
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the mean bone loss in width was 16 1.41% in the PRP group and 24 1.41% in the control group (Fig. 5). At 1 year after surgery, the mean bone loss was 26.5 0.71% in the PRP group and 35.5 2.12% in the control group, compared to the width at 1 month. The mean bone loss of the width in PRP group was significantly lower than that in the control group for both periods. The mean bone loss of the height of regenerated bone did not differ between the two groups at 1 week, 1 month or 6 months (Fig. 6). At 1 year after surgery, the mean of bone loss of height in the PRP group (1.42 0.18%) was significantly lower than that in the control group (2.09 0.36%), compared to the 1 week value. 4. Discussion
Fig. 4. Change in relative density of bone grafts over time. At 1 week, the relative density of the bone grafts in the PRP group was lower than that in the controls (*P < 0.05). However, there was no difference between the two groups after 1 month.
Fig. 5. The mean bone loss (%) in the width of regenerated bone. The mean bone loss (%) in the width of regenerated bone in the PRP group was lower than that in the controls for each period (*P < 0.05).
Fig. 6. The mean bone loss (%) in the height of regenerated bone. The mean bone loss (%) in the height of regenerated bone in the PRP group was lower than in the controls at 1 year (*P < 0.05).
relative density of bone grafts in the PRP group (1.50 0.22 mmAl equivalent) was lower than that in the control group (2.02 0.33 mmAl equivalent). The relative density of bone grafts in the PRP group then gradually increased to the same level as the controls from 1 month to 1 year after surgery (Fig. 4). At 6 months after surgery, and compared to the width at 1 month after surgery,
In the present study we studied the effectiveness of PRP in autologous cancellous bone and marrow grafting of cleft patients by radiographically evaluating the quality and quantity of bone formation in graft sites. In the qualitative analysis, the bone density of the bone graft with PRP was lower than that in the control group because bone fragments are more homogeneously amassed within the dense fibrin network of the PRP and thus the bone density would be diluted by the fibrin gel. The increase in bone density in the PRP group by 1 month to the same level as the controls suggests that the addition of PRP enhances new bone formation in the early postoperative period. The results of the quantitative analysis of the graft sites show that the mean bone loss in the PRP group was lower than that in the control group at 1 year after surgery. It appears that the resorption of regenerated bone that occurs postoperatively may be reduced significantly by using PRP. We believe that the fibrin networks of PRP might aid the decrease in postoperative bone resorption. The function of fibrin networks is as an osteoconductive scaffold (Soffer et al., 2003; Le Guehennec et al., 2005; Segura-Castillo et al., 2005; Catelas et al., 2006; Huh et al., 2006; Tajima et al., 2007), and thus the fibrin gel would have provided a matrix for cell growth and differentiation by enhancing three-dimensional intercellular interactions or cell adhesion, both of which are thought to be good environments for the maturation of osteoblasts (Catelas et al., 2006; Tajima et al., 2007). Another possible reason why PRP reduces bone resorption is that PRP accelerates wound healing in soft tissue. In the present study, minor wound dehiscence with bone exposure developed postoperatively in 2 patients in the control group (28.6%) and 1 patient in the PRP group (7.1%). The PRP gel not only provides haemostatic adhesion properties but also supplies the wound with valuable growth factors that enhance and promote the healing process (Petrungaro, 2001a,b; Kimura et al., 2005; Eppley et al., 2006). The growth factors in the graft sites may also increase immunomodulatory activity, which enhances the wound healing process. This acceleration of wound healing by PRP may result in a reduction in bone resorption. Marx et al. reported that PRP accelerated the rate and degree of bone formation in a bone graft. However, in our in vitro study (Tajima et al., 2007), when soluble factors from PRP were added to cultures of rat bone marrow stromal cells, they promoted proliferation but inhibited osteoblast differentiation in a dose-dependent manner. This finding agrees with those of other authors (Arpornmaeklong et al., 2004; Soffer et al., 2004; Ogino et al., 2005). Both TGF-b and PDGF have been reported to inhibit osteoblast differentiation and stimulate proliferation of cells in vitro, although the effect of TGF-b on osteoblast differentiation depends on the dose or the stage of differentiation of the osteoblast lineage (Kasperk et al., 1990; Marden et al., 1993; Harris et al., 1994; Cassiede et al., 1996; Hsieh and Graves, 1998; Lu et al., 2001; Lieb et al., 2004). The mean concentrations of platelets, TGF-b1, VEGF and PDGF-AB were all higher in the PRP used
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in the present study. The bone density in the PRP group was lower than that in the control group at 1 week; however between 1 month and 1 year, the level was the same between the two groups. The addition of PRP did not inhibit osteoblastic differentiation, probably because the growth factors in the PRP would have been diluted by body fluid circulation soon after surgery. In the present study we used autologous cancellous bone and marrow grafts from the iliac crest. PRP is not osteoinductive and must be used in combination with living bone cells. It is unlikely to significantly promote bone substitutes and other non-cellular graft materials such as HA (hydroxyapatite) and b-TCP (b-tricalcium phosphate). The environment in which PRP is applied is important, and the PRP may be either effective or inhibitory according to the concentrations of growth factors and the stage of bone formation. Our preparation system of PRP does not require a special PRP kit and is thus cost-effective. To start gelation, 2% calcium chloride was mixed with the PRP without thrombin. Many authors have used bovine thrombin and human thrombin, the use of which has been implicated in potentially life-threatening coagulopathies through the formation of cross-reactive antibodies to thrombin. To address this issue, autologous human thrombin or other coagulation promoters may be substituted. However, thrombin was not required in the present study to promote gelation. A gel consistency was obtained by mixing PRP gel (containing higher-than-serum concentrations of platelet-released growth factors) with the bone graft, producing a safe and cost-effective mixture. 5. Conclusion The addition of PRP to autogenous cancellous bone grafts appears to significantly reduce postoperative bone resorption after 1 year, PRP may preserve the width and height of the graft better than control, making autogenous cancellous bone grafting with PRP useful for alveolar bone grafting in cleft patients. References Aghaloo TL, Moy PK, Freymiller EG: Investigation of platelet-rich plasma in rabbit cranial defects: a pilot study. J Oral Maxillofac Surg 60: 1176e1181, 2002 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with anorganic bovine bone in the rabbit cranium: a pilot study. Int J Oral Maxillofac Implants 19: 59e65, 2004 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. A pilot study. Clin Oral Implants Res 16: 250e257, 2005 Arpornmaeklong P, Kochel M, Depprich R, Kubler NR, Wurzler KK: Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int J Oral Maxillofac Surg 33: 60e70, 2004 Boyne PJ, Sands NR: Secondary bone grafting of residual alveolar and palatal clefts. J Oral Surg 30: 87e92, 1972 Cassiede P, Dennis JE, Ma F, Caplan AI: Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro. J Bone Miner Res 11: 1264e1273, 1996 Catelas I, Sese N, Wu BM, Dunn JC, Helgerson S, Tawil B: Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Eng 12: 2385e2396, 2006 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 33: 56e59, 2004 Eppley BL, Woodell JE, Higgins J: Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg 114: 1502e1508, 2004 Eppley BL, Pietrzak WS, Blanton M: Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg 118: 147ee159e, 2006 Frechette JP, Martineau I, Gagnon G: Platelet-rich plasmas: growth factor content and roles in wound healing. J Dent Res 84: 434e439, 2005 Graziani F, Ducci F, Tonelli M, El Askary AS, Monier M, Gabriele M: Maxillary sinus augmentation with platelet-rich plasma and fibrinogen cryoprecipitate: a tomographic pilot study. Implant Dent 14: 63e69, 2005 Harris SE, Bonewald LF, Harris MA, Sabatini M, Dallas S, Feng JQ, GhoshChoudhury N, Wozney J, Mundy GR: Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in
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Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Reduction of bone resorption by the application of platelet-rich plasma (PRP) in bone grafting of the alveolar cleft Eriko Marukawa*, Hidekazu Oshina, Gaichi Iino, Keiichi Morita, Ken Omura Oral and Maxillofacial Surgery, Department of Oral Restitution, Division of Oral Health Sciences, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 9 December 2009 Accepted 23 April 2010
Objective: We evaluated the effectiveness of platelet-rich plasma (PRP) on the regeneration of autogenous cancellous bone and marrow grafted in the alveolar cleft. Design: Twenty patients with alveolar clefts were examined; 6 were the control group and received cancellous bone and marrow grafts without PRP, while the remaining 14 comprised the PRP group and received grafts with PRP. Prior to surgery, 50 ml of blood was withdrawn and 5 ml of PRP gel produced through centrifugal separation. The bone graft mixed with PRP was then packed into the alveolar cleft. Postoperative bone density was assessed as the aluminium-equivalent value on occlusal X-ray films in a qualitative analysis. Quantitative evaluation of regenerated bone was made with computed tomography and panoramic radiographs at 1 month, 6 months and 1 year after surgery. Results: Satisfactory bone bridging formation was observed in all patients without any complications. The bone density of the PRP group was lower than that of the control group at 1 week, but the same after 1 month. The added PRP reduced the resorption of regenerated bone postoperatively. Conclusion: Autogenous cancellous bone grafting with PRP, which significantly reduces postoperative bone resorption, is a reliable technique for alveolar bone grafting of cleft patients. Ó 2010 European Association for Cranio-Maxillo-Facial Surgery.
Keywords: Platelet-rich plasma (PRP) Alveolar bone grafting Alveolar cleft
1. Introduction In cleft lip and palate patients, bone grafting of the alveolar cleft is necessary for rehabilitation of oral function (Boyne and Sands, 1972; Witsenburg, 1985; Schendel, 1994; Keese and Schmelzle, 1995; Long et al., 1995). Among the available graft materials, autogenous bone is currently the preferred material. In some cleft patients it is difficult to augment the bone defect adequately because of the width of the gap between the maxillary segments, or as a result of bone resorption (Boyne and Sands,1972; Long et al.,1995; Opitz et al.,1999). A method for accelerating the speed of bone formation and reducing bone resorption in alveolar cleft bone grafting has been sought for some time. In 1998, platelet-rich plasma (PRP) was reported by Marx et al. (1998) to promote new bone formation in mandibular continuity defects and to cause faster maturation of autologous bone grafts. PRP contains a high concentration of platelets and is an autologous source of PDGF (platelet-derived growth factor), TGF-b, (transforming
* Corresponding author. Tel.: þ81 3 5803 5506; fax: þ81 3 5803 0199. E-mail address: [email protected] (E. Marukawa).
growth factor-b) and VEGF (vascular endothelial growth factor). Many reports (Marx et al., 1998; Aghaloo et al., 2002; Wiltfang et al., 2003; Aghaloo et al., 2004; Merkx et al., 2004; Schlegel et al., 2004; Graziani et al., 2005; Hokugo et al., 2005) have confirmed the effectiveness of PRP in enhancing bone regeneration in autologous bone grafts and other bone substitutes, although others have shown no benefit of PRP on bone formation (Choi et al., 2004; Wiltfang et al., 2004; Aghaloo et al., 2005; Pryor et al., 2005a,b; Raghoebar et al., 2005; Klongnoi et al., 2006a,b; Plachokova et al., 2006; Sarkar et al., 2006; Thorwarth et al., 2006). In this clinical study, we used autogenous cancellous bone with PRP in alveolar bone grafting of cleft patients in order to evaluate the effectiveness of PRP.
2. Materials and methods 2.1. Patients 20 non-syndromic patients with unilateral clefts treated with alveolar bone grafting during the period between March 2002 and March 2004 were included in this study. The patients were randomly assigned to two groups: the PRP group, who received
1010-5182/$ e see front matter Ó 2010 European Association for Cranio-Maxillo-Facial Surgery. doi:10.1016/j.jcms.2010.04.017
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autogenous cancellous bone and marrow grafts with PRP (14 patients; 7 males and 7 females, 11 unilateral cleft lip and palate and 3 cleft lip and alveolus); and the control group, who received grafts without PRP (6 patients; 2 males and 4 females, 1 unilateral cleft lip and palate and 5 cleft lip and alveolus). At the time of surgery the mean age of the patients was 20 4.7 years with a range 10e29 years. All procedures and materials were approved by the local Ethics Committee. Informed consent was obtained from all patients. 2.2. Preparation of PRP Prior to surgery 50 ml of blood was withdrawn from each patient with 5 ml of citrate phosphate dextrose (CPD: TERUMO, Japan) for anticoagulation. PRP gel was produced through centrifugal separation of whole blood. After the first centrifugation (800 g, 5 min), the blood was separated into plasma and red blood cells. The red blood cells were removed, and after a further centrifugation (1500 g, 5 min) of the remaining plasma, the bottom layer, which was rich in platelets and constituted approximately 10% of the total withdrawn blood volume, was collected for use as PRP. At the time of grafting, the solution was mixed with 2% calcium chloride at a volume ratio of 7 to 1 to start gelation and promote the release of growth factors, such as TGF-b, PDGFs, VEGF, and insulin-like growth factors (IGFs), from the platelets (Slater et al., 1995; Rendu and Brohard-Bohn, 2001; Eppley et al., 2004; Frechette et al., 2005). The TGF-b1, PDGF-AB and VEGF concentrations of each PRP preparation and whole blood were assayed using an ELISA (enzyme-linked immunosorbent assay) kit (Quantikaine, R&D Systems Inc., Minneapolis, MN, USA). 2.3. Surgical procedure Alveolar bone grafting was performed under general endotracheal anaesthesia. Incisions were made and gingival mucoperiosteal flaps were elevated in the standard fashion for alveolar bone grafting. Autogenous cancellous bone and marrow from the anterior iliac crest was used for each graft. The PRP gel was mixed with the bone graft to obtain a gel-like consistency (Fig. 1) and this mixture was then packed into the alveolar cleft and closed with gingival mucoperiosteal flaps. 2.4. Clinical and radiological evaluation Clinical and radiological follow-up examinations were carried out for 1 year. The quantity and quality of bone at the grafted sites were radiographically evaluated with the use of panoramic and occlusal radiographs and computed tomography (CT). Axial CT scans were obtained at a slice thickness of 1 mm. The level and window of each slice were optimally set to allow precise delineation between bone and soft tissue. Intra-oral radiographs of the grafted sites were taken to document progressive bone formation every month after surgery. We assessed the bone density of the grafted sites on occlusal X-ray films in a qualitative analysis. The measurement procedure consisted of image acquisition, correction for radiographic variation using an aluminium wedge (Fig. 2a) and transformation of the grey values into aluminium-equivalent values to produce the relative density of the bone. All relative density was subtracted from the pre-operative aluminium-equivalent values to correct for variables such as soft tissue density that can be confounding factors. Quantitative evaluation of the regenerated bone was made using the CT scans (Fig. 2b) and panoramic radiographs (Fig. 2c). The width of the alveolar crest was measured using a CT scan at 1 month, 6 months and 1 year after surgery. The width of grafted
Fig. 1. The application of PRP to autogenous bone particles. The cancellous marrow bone particles were mixed with the PRP for gel formation and activation of the PRP. They can be handled easily.
sites at 1 month was set as the baseline value, and the ratio of bone loss at 6 months and 1 year after surgery calculated. Using panoramic radiographs, the height of the alveolar crest was measured at 1 week, 1 month, 6 months and 1 year after surgery. The height of grafted sites at 1 week was set as the baseline value and the ratio of bone loss at 1 month, 6 months and 1 year after surgery calculated. The data for all the patients was obtained and analysed by two researchers who were blinded as to which treatment the patients had received. The average of the two sets of measurements for each patient was used for comparison of the two groups. The ManneWhitney U test revealed a statistical significant difference (P < 0.05) between the groups of PRP patients and controls.
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Fig. 2. Evaluation of the quantity and quality in grafted sites. (a) An aluminium step wedge was placed during all occlusal radiographic exposures. The thickness of the alveolar bone at each pixel was calculated as an equivalent aluminium thickness. (b) The width of the alveolar crest was measured on axial CT images. The mean bone loss (%) at 6 months and 1 year, compared to 1 month, was evaluated. (c) The height of the alveolar crest was measured on panoramic radiographs. The mean bone loss (%) at 1 month, 6 months and 1 year, compared to 1 week, was evaluated.
Fig. 3. (a) The concentrations of platelets in serum and PRP. (b) The concentrations of TGF-b1 in serum and PRP. (c) The concentrations of VEGF in serum and PRP. (d) The concentrations of PDGF-AB in serum and PRP. Platelet counts in PRP were about 3-fold greater, and growth factors were 2e3-fold greater, than in serum.
3. Results 3.1. Clinical findings The mean concentrations of platelets, TGF-b1, VEGF and PDGFAB were all higher in the PRP (Fig. 3aed), with platelets being about 3-fold higher, and platelet-released growth factors about 2e3-fold higher, than in serum. Of the 20 patients, 17 (control group, 4; PRP group, 13) had an uneventful course postoperatively. In the 3 remaining patients
(control group, 2; PRP group, 1), wound dehiscence developed with minor bone exposure. However, these exposures closed during the follow-up period. No other complications were observed. Bone formation was satisfactory in all patients. 3.2. Radiological evaluation The mean relative density of bone grafts aluminium-equivalent values (mmAl equivalent) and mean total bone loss in the regenerated bone are shown in Figs. 4e6. At 1 week after surgery, the
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the mean bone loss in width was 16 1.41% in the PRP group and 24 1.41% in the control group (Fig. 5). At 1 year after surgery, the mean bone loss was 26.5 0.71% in the PRP group and 35.5 2.12% in the control group, compared to the width at 1 month. The mean bone loss of the width in PRP group was significantly lower than that in the control group for both periods. The mean bone loss of the height of regenerated bone did not differ between the two groups at 1 week, 1 month or 6 months (Fig. 6). At 1 year after surgery, the mean of bone loss of height in the PRP group (1.42 0.18%) was significantly lower than that in the control group (2.09 0.36%), compared to the 1 week value. 4. Discussion
Fig. 4. Change in relative density of bone grafts over time. At 1 week, the relative density of the bone grafts in the PRP group was lower than that in the controls (*P < 0.05). However, there was no difference between the two groups after 1 month.
Fig. 5. The mean bone loss (%) in the width of regenerated bone. The mean bone loss (%) in the width of regenerated bone in the PRP group was lower than that in the controls for each period (*P < 0.05).
Fig. 6. The mean bone loss (%) in the height of regenerated bone. The mean bone loss (%) in the height of regenerated bone in the PRP group was lower than in the controls at 1 year (*P < 0.05).
relative density of bone grafts in the PRP group (1.50 0.22 mmAl equivalent) was lower than that in the control group (2.02 0.33 mmAl equivalent). The relative density of bone grafts in the PRP group then gradually increased to the same level as the controls from 1 month to 1 year after surgery (Fig. 4). At 6 months after surgery, and compared to the width at 1 month after surgery,
In the present study we studied the effectiveness of PRP in autologous cancellous bone and marrow grafting of cleft patients by radiographically evaluating the quality and quantity of bone formation in graft sites. In the qualitative analysis, the bone density of the bone graft with PRP was lower than that in the control group because bone fragments are more homogeneously amassed within the dense fibrin network of the PRP and thus the bone density would be diluted by the fibrin gel. The increase in bone density in the PRP group by 1 month to the same level as the controls suggests that the addition of PRP enhances new bone formation in the early postoperative period. The results of the quantitative analysis of the graft sites show that the mean bone loss in the PRP group was lower than that in the control group at 1 year after surgery. It appears that the resorption of regenerated bone that occurs postoperatively may be reduced significantly by using PRP. We believe that the fibrin networks of PRP might aid the decrease in postoperative bone resorption. The function of fibrin networks is as an osteoconductive scaffold (Soffer et al., 2003; Le Guehennec et al., 2005; Segura-Castillo et al., 2005; Catelas et al., 2006; Huh et al., 2006; Tajima et al., 2007), and thus the fibrin gel would have provided a matrix for cell growth and differentiation by enhancing three-dimensional intercellular interactions or cell adhesion, both of which are thought to be good environments for the maturation of osteoblasts (Catelas et al., 2006; Tajima et al., 2007). Another possible reason why PRP reduces bone resorption is that PRP accelerates wound healing in soft tissue. In the present study, minor wound dehiscence with bone exposure developed postoperatively in 2 patients in the control group (28.6%) and 1 patient in the PRP group (7.1%). The PRP gel not only provides haemostatic adhesion properties but also supplies the wound with valuable growth factors that enhance and promote the healing process (Petrungaro, 2001a,b; Kimura et al., 2005; Eppley et al., 2006). The growth factors in the graft sites may also increase immunomodulatory activity, which enhances the wound healing process. This acceleration of wound healing by PRP may result in a reduction in bone resorption. Marx et al. reported that PRP accelerated the rate and degree of bone formation in a bone graft. However, in our in vitro study (Tajima et al., 2007), when soluble factors from PRP were added to cultures of rat bone marrow stromal cells, they promoted proliferation but inhibited osteoblast differentiation in a dose-dependent manner. This finding agrees with those of other authors (Arpornmaeklong et al., 2004; Soffer et al., 2004; Ogino et al., 2005). Both TGF-b and PDGF have been reported to inhibit osteoblast differentiation and stimulate proliferation of cells in vitro, although the effect of TGF-b on osteoblast differentiation depends on the dose or the stage of differentiation of the osteoblast lineage (Kasperk et al., 1990; Marden et al., 1993; Harris et al., 1994; Cassiede et al., 1996; Hsieh and Graves, 1998; Lu et al., 2001; Lieb et al., 2004). The mean concentrations of platelets, TGF-b1, VEGF and PDGF-AB were all higher in the PRP used
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in the present study. The bone density in the PRP group was lower than that in the control group at 1 week; however between 1 month and 1 year, the level was the same between the two groups. The addition of PRP did not inhibit osteoblastic differentiation, probably because the growth factors in the PRP would have been diluted by body fluid circulation soon after surgery. In the present study we used autologous cancellous bone and marrow grafts from the iliac crest. PRP is not osteoinductive and must be used in combination with living bone cells. It is unlikely to significantly promote bone substitutes and other non-cellular graft materials such as HA (hydroxyapatite) and b-TCP (b-tricalcium phosphate). The environment in which PRP is applied is important, and the PRP may be either effective or inhibitory according to the concentrations of growth factors and the stage of bone formation. Our preparation system of PRP does not require a special PRP kit and is thus cost-effective. To start gelation, 2% calcium chloride was mixed with the PRP without thrombin. Many authors have used bovine thrombin and human thrombin, the use of which has been implicated in potentially life-threatening coagulopathies through the formation of cross-reactive antibodies to thrombin. To address this issue, autologous human thrombin or other coagulation promoters may be substituted. However, thrombin was not required in the present study to promote gelation. A gel consistency was obtained by mixing PRP gel (containing higher-than-serum concentrations of platelet-released growth factors) with the bone graft, producing a safe and cost-effective mixture. 5. Conclusion The addition of PRP to autogenous cancellous bone grafts appears to significantly reduce postoperative bone resorption after 1 year, PRP may preserve the width and height of the graft better than control, making autogenous cancellous bone grafting with PRP useful for alveolar bone grafting in cleft patients. References Aghaloo TL, Moy PK, Freymiller EG: Investigation of platelet-rich plasma in rabbit cranial defects: a pilot study. J Oral Maxillofac Surg 60: 1176e1181, 2002 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with anorganic bovine bone in the rabbit cranium: a pilot study. Int J Oral Maxillofac Implants 19: 59e65, 2004 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. A pilot study. Clin Oral Implants Res 16: 250e257, 2005 Arpornmaeklong P, Kochel M, Depprich R, Kubler NR, Wurzler KK: Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int J Oral Maxillofac Surg 33: 60e70, 2004 Boyne PJ, Sands NR: Secondary bone grafting of residual alveolar and palatal clefts. J Oral Surg 30: 87e92, 1972 Cassiede P, Dennis JE, Ma F, Caplan AI: Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro. J Bone Miner Res 11: 1264e1273, 1996 Catelas I, Sese N, Wu BM, Dunn JC, Helgerson S, Tawil B: Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Eng 12: 2385e2396, 2006 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 33: 56e59, 2004 Eppley BL, Woodell JE, Higgins J: Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg 114: 1502e1508, 2004 Eppley BL, Pietrzak WS, Blanton M: Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg 118: 147ee159e, 2006 Frechette JP, Martineau I, Gagnon G: Platelet-rich plasmas: growth factor content and roles in wound healing. J Dent Res 84: 434e439, 2005 Graziani F, Ducci F, Tonelli M, El Askary AS, Monier M, Gabriele M: Maxillary sinus augmentation with platelet-rich plasma and fibrinogen cryoprecipitate: a tomographic pilot study. Implant Dent 14: 63e69, 2005 Harris SE, Bonewald LF, Harris MA, Sabatini M, Dallas S, Feng JQ, GhoshChoudhury N, Wozney J, Mundy GR: Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in
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