- Email: [email protected]
Effects of Platelet-Rich Plasma at the Cellular Level on Healing of Autologous Bone-Grafted Mandibular Defects in Dogs
J Oral Maxillofac Surg 65:721-727, 2007
Effects of Platelet-Rich Plasma at the Cellular Level on Healing of Autologous Bone-Grafted Mandibular Defects in Dogs David Gerard, PhD,* Eric R. Carlson, DMD, MD,† Jack E. Gotcher, DMD, PhD,‡ and Mykle Jacobs, DDS§ Purpose: This study describes the effect of platelet-rich plasma (PRP) at the cellular level on immediate
autologous bone grafts in dog mandibles. Materials and Methods: Twelve adult dogs weighing 40 to 50 pounds received bilateral inferior mandibular border resections measuring 2 cm ⫻ 1 cm. The right side was grafted with milled autologous iliac corticocancellous bone along with 2 cc of PRP taken from the same animal. The left side had the same amount of milled bone placed in the defect without PRP. Three animals were sacrificed at 1, 2, 3, and 6 months postsurgery. Ten and 3 days before sacrifice, all dogs received 10 mg/kg body weight of intravenous tetracycline. At sacrifice, the grafts along with adjacent native bone were harvested and immediately fixed in Carson’s fixative for 48 to 72 hours. The samples were then dehydrated over a 2-week period in a graded ethanol series and embedded in Spurr’s plastic. Two 100- sections from the center of each graft were cut, mounted on glass slides, ground to 40 , and stained. A digitally generated grid was superimposed over each section, to give 32 fields of 2.5 mm2. Each of these fields was examined at a magnification of ⫻100 to determine the number of osteoblasts and osteoclasts present. Results: The mean average of the total numbers of osteoblasts and osteoclasts were significantly higher in the PRP graft sites than in the non-PRP graft sites at 1 month. However, if specific fields were compared, then 14 of the 32 fields showed no difference in the number of osteoblasts and osteoclasts. At 2, 3, and 6 months, there was no significant difference in the total number of osteoblasts or osteoclasts in the PRP and non-PRP grafts, or in any of the 32 fields. Conclusions: At the cellular level, PRP increased the number of osteoblasts and osteoclasts recruited to the graft site at 1 month, and this overall increase was more evident at the superior and lateral margins of the graft than in other areas. Fields along the inferior margin showed the fewest number of cells for both the PRP and non-PRP grafts. At later times there was no significant difference in the number of osteoblasts and osteoclasts in the PRP and non-PRP graft sites in any region of the grafts. This study indicates that the increased number of osteoblasts and osteoclasts in the graft sites due to the addition of PRP was short-lived, and that autologous bone grafts without PRP had similar numbers of active bone cells after 1 month in this animal model. © 2007 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 65:721-727, 2007 The use of fresh, healthy autologous corticocancellous bone remains the gold standard for bone graft reconstruction of the jaws. Many other materials have been used to repair bony defects, but their efficacy is still measured by how they compare with autologous grafts.
Researchers have looked for ways to enhance xenograft materials by various means, including the addition of autologous bone,1 the addition of growth factors2-4 or the use of guided tissue regeneration membranes.5,6 However, if the patient is not compro-
Received from the Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of Medicine, Knoxville, TN. *Associate Professor, Director of Research. †Professor and Chairman. ‡Professor. §Formerly, Chief Resident; Currently, Fellow, Oral and Maxillofacial Surgery, St John’s Mercy Medical Center, St Louis, MO. Presented in part at the American Association of Oral and Max-
illofacial Surgeons’ 87th Annual Meeting and Scientific Sessions. Address correspondence and reprint requests to Dr Carlson: University of Tennessee Medical Center, Department of Oral and Maxillofacial Surgery, 1930 Alcoa Highway, Suite 335, Knoxville, TN 37920; e-mail: [email protected] © 2007 American Association of Oral and Maxillofacial Surgeons
0278-2391/07/6504-0020$32.00/0 doi:10.1016/j.joms.2006.09.025
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722 mised, then autologous bone grafts are still the most commonly used material for treating bony defects, especially of the craniofacial region. In recent years, researchers have looked for ways to enhance healing of autologous bone grafts by adding individual growth factors3 or PRP.7-12 The rationale behind using PRP is that activation of a large number of platelets at the time of graft placement releases a bolus of various growth factors,13 including platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, vascular endothelial growth factor (VEGF), insulinlike growth factor (IGF)-I, and epidermal growth factor (EGF).14 This increased concentration of growth factors is thought to have a positive effect on the recruitment of osteoblasts (ob) and osteoclasts (oc) precursors, differentiation and activation of osteoblasts and osteoclasts, and recruitment and activation of endothelial cells, enhancing angiogenesis. Autologous bone grafts heal through a mechanism whereby the body replaces the nonviable grafted bone with viable new bone. In a previous study, we reported on this process in a dog mandible bone graft model at the tissue level using histomorphometry.15 In that study, we described the effect of PRP on graft healing at 1, 2, 3, and 6 months postsurgery. We found that PRP increased the amount of new bone formed and the amount of nonviable grafted bone removed up to 2 months. After 2 months, there was no difference between the PRP and non-PRP graft sites. At the cellular level, many researchers, notably Frost,16 have studied osteoblast and osteoclast activity and morphology in normal bone. In addition, osteoblast and osteoclast activity and morphology have been described for some skeletal diseases, including Paget’s disease,16 osteoporosis,17 and osteopetrosis.18,19 To the best of our knowledge, there are no published studies examining bone graft healing at the cellular level. Because osteoblasts and osteoclasts are doing the work of bone formation and resorption, it is important to understand the effect of PRP on these cells. The hypothesis of this study is that the addition of PRP will increase the number of osteoblasts and osteoclasts in autologous mandibular bone grafts at 1 and 2 months, but not at 3 and 6 months. This hypothesis is based on the results of our previous study that showed increased formation of new bone and resorption of nonviable bone at the tissue level after 1 month and 2 months.15 Quantitative analysis of cell numbers was performed on stained sections. Our findings yield valuable information on the effect of PRP at the cellular level on autologous mandibular bone grafts. They show that the influence of PRP on early bone graft healing is localized to certain defined regions of the grafts in this animal model.
PLATELET-RICH PLASMA AND GRAFT HEALING
Materials and Methods PRP PREPARATION
PRP was prepared using a Harvest Technologies Smart Prep Centrifuge, with the PRP-20cc prep kit (Harvest Technologies, Plymouth, MA). A 20-cc sample of peripheral blood was drawn from each animal and processed for PRP preparation using the manufacturer’s instructions. An additional 2 cc of blood was drawn at the same time to calculate peripheral platelet counts for each animal. SURGICAL PROCEDURE
The surgical procedure for donor site bone harvest and recipient tissue site preparation and grafting was standardized and reported in a previous publication.15 TISSUE PROCESSING
Three animals were killed at 1, 2, 3, and 6 months postsurgery. After euthanasia, the mandibular defects and adjacent mandible (2 to 3 mm beyond the screws) were harvested intact and immediately placed into Carson’s fixative. Within the first 24 hours, standardized radiographs of the samples were obtained. After 48 hours of fixation, the undecalcified samples were dehydrated in an ethanol series over a 2-week period and embedded in Spurr’s plastic. Serial longitudinal 100- sections were cut through the graft sites and adjacent mandible with a Leitz 1600 bone saw (Leitz, Oberkochen, Germany). QUANTITATIVE CELL COUNT ANALYSIS
Two sections from the center of each defect were glued onto petrographic slides and ground to a thickness of 40 . The sections were stained with alizarine red, Sanderson’s rapid bone stain, and/or light green stain. The slides were examined at ⫻100, and a grid of 32 equal-sized fields of 2.5 mm2 was superimposed over the graft sites using Image-Pro Express software (Media Cybernetics, Silver Spring, MD) interfaced with a Leica DMRX research microscope (Leica Microsystems, Wetzlar, Germany) and a Sony digital camera (Sony, Tokyo, Japan) (Fig 1A). An image of each field was digitally captured. The area within each field was then examined at higher magnification (⫻250) to accurately identify and count all cells within the field. Thus 64 fields (32 from each section) were examined for each graft site, and 192 fields (64 from each of 3 animals) were examined for each time and treatment (PRP or non-PRP). The total number of osteoblasts (Fig 2) and osteoclasts (Fig 3) from each section, as well as from each field, was recorded.
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FIGURE 1. A, This schematic drawing depicts the 32-field grid overlay on a section of the 1 cm ⫻ 2 cm graft site. The fields are numbered sequentially from anterior to posterior and superior to inferior. Each field is 2.5 mm2. B, This figure shows the mean average of the number of osteoblasts (ob) and osteoclasts (oc) in each of the 32 fields analyzed at 1 month for both the PRP and non-PRP grafts. The numbers in red indicate a significant difference in the number of osteoblasts or osteoclasts counted in those fields. P ⬍ .05. Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
STATISTICAL ANALYSIS
The number of osteoblasts and osteoclasts counted for each section was calculated for each time and treatment (n ⫽ 6, 2 sections from each graft site and 3 animals each time). The mean average of the total number of osteoblasts and osteoclasts for PRP and non-PRP grafts were compared for each time using 1-way analysis of variance (ANOVA) (Figs 4, 5). Also, the mean averages of osteoblasts and osteoclasts for each of the corresponding 32 fields were compared using 1-way ANOVA. For example, the mean average of the number of osteoblasts in field 1 was compared for
the PRP and non-PRP grafts for each time (n ⫽ 6, field 1 from 2 sections and 3 animals for each time point and treatment). Differences in the number of osteoblasts or osteoclasts in the PRP and non-PRP grafts sites were considered significant at P ⬍ .05.
Results PRP PREPARATION
Platelet concentration averaged 388% for 1-month animals, 344% for 2-month animals, 386% for 3-month animals, and 334% for 6-month animals.15 Two ani-
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PLATELET-RICH PLASMA AND GRAFT HEALING
FIGURE 4. Comparison of the mean average number of osteoblasts in PRP and non-PRP grafts for 1, 2, 3, and 6 months. *Indicates a significant difference, P ⬍ .05. Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
FIGURE 2. This photograph shows numerous active osteoblasts on the forming surface of new bone. This section was from a non-PRP graft at 3 months stained with Sanderson’s rapid bone stain and light-green stain. (Original magnification ⫻250.) Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
mals at the 3-month time point did not have concentrations calculated. These platelet concentrations are comparable to those described by Marx and coworkers13,14 in their clinical trials. QUANTITATIVE CELL COUNT ANALYSIS
Comparing the mean average of the total number of osteoblasts found in each section of the PRP and non-PRP grafts revealed a significantly higher number
in the PRP grafts at 1 month (P ⬍ .05). This was also true for osteoclasts (P ⬍ .05). However, comparing the total number of osteoblasts and osteoclasts in the grafts at 2, 3, and 6 months revealed no significant difference (Figs 4, 5). To further refine possible differences in cellular activity in different areas of the bone graft, comparisons were made between each field and compared for the PRP and non-PRP grafts. Thus, all numbered fields (eg, the fields marked “1,” located at the most anterior/superior region of the graft [Fig 1]), were compared. By analyzing and comparing only fields in the same location in the graft sites, a clearer picture of where PRP may most influence graft healing emerged. The results of this analysis are shown for the 1-month time point in Figure 1B. In 14 of the 32 fields that cover the graft site, there was no significant difference in the number of osteoblasts and osteoclasts between the PRP and non-PRP graft sites; in the other 18 fields, the difference in the number of osteoblasts and osteoclasts was significant. Generally, the fields along
FIGURE 3. This photograph shows 2 osteoclasts on the surface of a Howship’s lacunae at 3 months in a PRP graft. The section was stained with Sanderson’s rapid bone stain and light-green stain. (Original magnification ⫻250.)
FIGURE 5. This shows the mean average number of osteoclasts in PRP and non-PRP grafts for 1, 2, 3, and 6 months. *Indicates a significant difference, P ⬍ .05.
Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
GERARD ET AL
the anterior, posterior, and superior margins showed the greatest increase in the numbers of osteoblasts and osteoclasts when PRP was added to the graft. The effect of PRP was not significant along the inferior margin and toward the center of the graft site. Moreover, fewer osteoblasts and osteoclasts were counted along the inferior margin in both the PRP and non-PRP grafts; in some of these fields, very few osteoblasts and no osteoclasts were seen (Fig 1). At 2, 3, and 6 months, there was no statistically significant difference in the number of osteoblasts and osteoclasts with or without PRP in any of the 32 fields (Figs 4, 5).
Discussion This is the first study to report on the effect of PRP on autologous bone grafts at the cellular level. Using digital image capture techniques, it was possible to subdivide each graft section into 32 equal fields of 2.5 mm2 on slides of 2 central longitudinal sections of each graft. In this way, all osteoblasts and osteoclasts within each field were counted. Comparing the total number of osteoblasts and osteoclasts from all fields for the PRP and non-PRP grafts showed that the PRP grafts had significantly more osteoblasts and osteoclasts at 1 month, but there was no significant difference at 2, 3, or 6 months. Because the 32 fields were reproduced over each section, it was also possible to examine cell numbers for each field separate from the whole graft. In doing so, results were further refined to demonstrate that even at 1 month, not all areas of the graft showed a difference in the numbers of osteoblasts and osteoclasts. Thus, fields at the anterior and posterior margin and at the superior edge of the graft were most significantly affected by the addition of PRP. The fields at the center of the graft and at the inferior margin generally showed no difference in cell numbers with the addition of PRP. Factors that may contribute to an increased effect of PRP on the anterior and posterior regions of the graft include early angiogenesis in these regions due to high concentrations of EGF and PDGF that stimulate endothelial cell proliferation. In addition, increased concentrations of TGF- and IGF-I stimulate both osteoblast and osteoclast precursor cells to divide and differentiate. In the superior region of the graft, these growth factors, along with the presence of a robust neurovascular bundle in this animal model, probably stimulate early angiogenesis as well as osteoblast and osteoclast activity. The Utah paradigm stresses the important role of mechanical forces on bone healing.20 Removal of a significant portion of a weight-bearing bone will have a significant effect on bone activity by increasing the load on the areas adjacent to the graft. This effect will most likely be seen along the margins of the defect that are in con-
725 tact with native bone placed under an increased load. Although every effort was made to maintain the periosteum, its disruption during surgical placement of the grafts may have played a role in PRP’s lack of significant effect on the numbers of osteoblasts and osteoclasts along the inferior border of the graft. In addition, consolidation of the grafts in this region may have caused loss of bone from these areas. This possibility is supported by the fact that few osteoblasts or osteoclasts were found in many fields in this area in either the PRP or non-PRP grafts at 1 month. Osteoclast size and numbers of nuclei have been reported to be significant factors in determining the activity of these cells.21,22 In the present study, we found no observable difference in the size of osteoclasts or in the number of nuclei in these cells between the PRP and non-PRP grafts. This finding suggests that PRP did not affect the activity of the cells but only increased the number of osteoclasts at 1 month. In our earlier study,15 we reported a significant increase in new bone in the PRP grafts at 2 months, whereas in the present study we found no significant difference in the number of osteoblasts at 2 months. This difference can be explained by the fact that the higher number of osteoblasts at 1 month would contribute to greater new bone formation. This increase would be seen even at 2 months at the tissue level. Some studies have reported long-term positive effects of PRP on bone grafts. Marx et al13 first described the effect of PRP on mandibular bone grafts in a series of 84 patients. These authors reported that the addition of PRP significantly enhanced autologous bone grafts out to 6 months over non-PRP grafts. Wiltfang et al23 reported on 39 patients receiving sinus floor augmentation with either beta-tricalciumphosphate (beta-TCP) alone or with PRP. At 6 months postsurgery, the PRP-added grafts had 8% to 10% more new bone, but the PRP did not increase the rate at which the beta-TCP was resorbed. Kassolis and Reynolds24 reported on 10 patients who received bilateral maxillary subantral sinus augmentation, with 1 site receiving freeze-dried bone allograft (FDBA) plus PRP and the other side receiving FDBA with a resorbable membrane. At 4.5 and 6 months postsurgery, the PRP-treated grafts showed a higher percentage of vital tissue and bone formation and a smaller amount of FDBA, suggesting that the PRP enhanced both bone formation and the removal of FDBA. Lekovic et al25 reported on 52 grade II mandibular molar furcation defects treated with either open flap debridement or bovine porous bone mineral (BPBM) along with PRP and guided tissue regeneration (GTR) membrane. At 6 months posttreatment, the sites were evaluated clinically, and the BPBM/PRP/GTR sites showed better results. These authors stated the need
726 for further studies to determine what role each of the 3 components of the experimental treatment played in the improved results. Mazor et al26 reported on 105 patients who received sinus augmentation with a composite graft of 30% to 40% autogenous bone and 60% to 70% xenograft, along with PRP. Dental implants were placed at the time of augmentation and uncovered at 6 months, at which time all implants were osseointegrated. There were no controls for this study. A growing body of literature showing that PRP enhances graft healing early, but not long-term, supports our findings in the present study. Those earlier studies investigated the use of PRP in a number of different applications in conjunction with autologous bone grafts or with various allograft materials. For example, Hanna et al27 examined bovine-derived xenografts (BDX) with and without PRP in 13 patients with bilateral periodontal defects. At 6 months, there was no difference between the PRP and non-PRP sites with reference to a number of clinical measurements. No earlier time points were used for this study. Lekovic et al28 examined the used of BPMB either with PRP or with PRP plus GTR in 21 patients with bilateral periodontal defects and measured various clinical parameters at 6 months posttreatment. Both treatments showed significant improvement, but there was no significant difference between the 2 groups. A number of animal studies have investigated the efficacy of PRP in conjunction with various grafting materials at various times. Schiegel et al29 used a pig model to study the effect of PRP on autogenous grafts or a bovine collagen device using both microradiography and light microscopy. They found that PRP had a significant positive effect on autogenous grafts at 2 weeks, but did not enhance the bovine collagen device. At 12 weeks, there was no difference between the PRP and non-PRP treatment groups. Kovacs et al30 investigated the effect of PRP combined with betaTCP on mandibular defects in dogs using densitometric and histological techniques and found that PRP enhanced bone formation at 6 and 12 weeks. No later time points were examined in this study. Suba et al31 examined the healing of premolar extraction sockets in dogs when Cerasorb or Cerasorb with PRP were placed in the sockets. At 6 and 12 weeks, more new bone was found in the PRP sites, but at 24 weeks, there was no difference between the PRP and nonPRP sites. Grageda et al32 compared the use of 2 allograft materials with and without PRP in sinus augmentation in sheep. Histomorphometric analysis indicated that PRP did not enhance bone formation at either 3 or 6 months. No earlier time points were studied. Numerous other studies have examined the effect of PRP on different bony defects in animal models.
PLATELET-RICH PLASMA AND GRAFT HEALING
Some reported that PRP is effective in enhancing bone healing only early in the process,33-36 whereas others reported that PRP has no beneficial effect at any time studied.37-43 The present study further defines the role of PRP in the early enhancement of autologous bone grafts in the canine mandible. It appears that PRP enhances the number of osteoblasts and osteoclasts in the graft site only at 1 month. At 2, 3, and 6 months, no significant difference in the total number of osteoblasts and osteoclasts was seen in the PRP and nonPRP graft sites. Using digital imaging analysis of reproducible sites in each graft, we further determined that the enhancement of PRP was restricted to peripheral areas of the graft site that were in contact with native bone, and that this enhancement did not persist beyond 1 month. The results of the present study support a growing body of literature indicating that the positive effects of PRP on bone grafts are seen only early in the healing process.
References 1. Mazor Z, Peleg M, Garg AK, et al: Platelet-rich plasma for bone enhancement in sinus floor augmentation with simultaneous implant placement: Patient series study. Implant Dent 13:65, 2004 2. Roldan JC, Jepsen S, Schmidt C, et al: Sinus floor augmentation with simultaneous placement of dental implants in the presence of platelet-rich plasma or recombinant human bone morphogenetic protein-7. Clin Oral Implants Res 15:716, 2004 3. Roldan JC, Jepsen S, Miller J, et al: Bone formation in the presence of platelet-rich plasma vs bone morphogenetic protein-7. Bone 34:80, 2004 4. Jung RE, Schmoekel HG, Zwahlen R, et al: Platelet-rich plasma and fibrin as delivery systems for recombinant human bone morphogenetic protein-2. Clin Oral Implants Res 16:676, 2005 5. Camargo PM, Lekovic V, Weinlaender M, et al: A reentry study on the use of bovine porous bone mineral, GTR, and plateletrich plasma in the regenerative treatment of intrabony defects in humans. Int J Periodontics Res Dent 25:49, 2005 6. Camargo PM, Lekovic V, Weinlaender M, et al: Platelet-rich plasma and bovine porous bone mineral combined with guided tissue regeneration in the treatment of intrabony defects in humans. J Periodontal Res 37:300, 2002 7. Fennis JP, Stoelinga PJ, Jansen JA: Mandibular reconstruction: A clinical and radiographic animal study on the use of autogenous scaffolds and platelet-rich plasma. Int J Oral Maxillofac Surg 31:281, 2002 8. Amet EM: Computerized tomography with CT models for contemporary ramus frame implant planning and construction. J Oral Implantol 24:152, 1998 9. Jakse N, Tangl S, Gilli R, et al: Influence of PRP on autogenous sinus grafts. An experimental study on sheep. Clin Oral Implants Res 14:578, 2003 10. Oyama T, Nishimoto S, Tsugawa T, et al: Efficacy of plateletrich plasma in alveolar bone grafting. J Oral Maxillofac Surg 62:555, 2004 11. Butterfield KJ, Bennett J, Gronowicz G, et al: Effect of plateletrich plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model. J Oral Maxillofac Surg 63:370, 2005 12. Raghoebar GM, Schortinghuis J, Liem RS, et al: Does plateletrich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor? Clin Oral Implants Res 16:349, 2005
GERARD ET AL 13. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:48, 1998 14. Marx RE: Platelet-rich plasma: Evidence to support its use. J Oral Maxillofac Surg 47:489, 2004 15. Gerard D, Carlson ER, Gotcher JE, et al: Effects of platelet-rich plasma on the healing of autologous bone grafted mandibular defects in dogs. J Oral Maxillofac Surg 64:443, 2006 16. Frost HM: Intermediary Organization of the Skeleton. Boca Raton, FL, CRC Press, 1986 17. Reddy SV, Menaa C, Singer FR, et al: Cell biology of Paget’s disease. J Bone Miner Res 14(Suppl 2):3, 1999 18. Gruber HE, Ivey JL, Thompson ER, et al: Osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis. Miner Electrolyte Metab 12:246, 1986 19. Henriksen K, Gram J, Schaller S, et al: Characterization of osteoclasts from patients harboring a G215R mutation in CIC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 164:1537, 2004 20. Marks SC Jr: Osteoclast biology: Lesions from mammalian mutations. Am J Med Genet 34:43, 1989 21. Frost HM: A 2003 update of bone physiology and Wolff’s law for clinicians. Angle Orthod 74:3, 2004 22. Lees RL, Heersche JN: Macrophage colony-stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size. J Bone Miner Res 14:937, 1999 23. Wiltfang J, Schlegel KA, Schultze-Mosgau S, et al: Sinus floor augmentation with beta-tricalciumphosphate (beta-TCP): Does platelet-rich plasma promote its osseous integration and degradation? Clin Oral Implants Res 14:213, 2003 24. Kassolis JD, Reynolds MA: Evaluation of the adjunctive benefits of platelet-rich plasma in subantral sinus augmentation. J Craniofac Surg 16:280, 2005 25. Lekovic V, Camargo PM, Weinlaender M, et al: Effectiveness of a combination of platelet-rich plasma, bovine porous bone mineral and guided tissue regeneration in the treatment of mandibular grade II molar furcations in humans. J Clin Periodontol 30:746, 2003 26. Mazor Z, Peleg M, Garg AK, et al: Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: Patient series study. Implant Dent 13:65, 2004 27. Hanna R, Trejo PM, Weltman RL: Treatment of intrabony defects with bovine-derived xenograft alone and in combination with platelet-rich plasma: A randomized clinical trial. J Periodontol 75:1668, 2004 28. Lekovic V, Camargo PM, Weinlaender M, et al: Comparison of platelet-rich plasma, bovine porous bone mineral, and guided tissue regeneration versus platelet-rich plasma and bovine porous bone mineral in the treatment of intrabony defects: A reentry study. J Periodontol 73:198, 2002
727 29. Schiegel KA, Donnath K, Rupprecht S, et al: De novo bone formation using bovine collagen and platelet-rich plasma. Biomaterials 25:5387, 2004 30. Kovacs K, Velich N, Huszar T, et al: Histomorphometric and densitometric evaluation of the effects of platelet-rich plasma on the remodeling of beta-tricalcium phosphate in beagle dogs. J Craniofac Surg 16:150, 2005 31. Suba Z, Takacs, Gyulai-Gaal S, et al: Facilitation of beta-tricalcium phosphate-induced alveolar bone regeneration by platelet-rich plasma in beagle dogs: A histologic and histomorphometric study. Int J Oral Maxillofac Implants 19:832, 2004 32. Grageda E, Lozada JL, Boyne PJ, et al: Bone formation in the maxillary sinus by using platelet-rich plasma: An experimental study in sheep. J Oral Implantol 31:2, 2005 33. Jakse N, Tangl S, Gilli R, et al: Influence of PRP on autogenous sinus grafts: An experimental study on sheep. Clin Oral Implants Res 14:578, 2003 34. Wiltfang J, Kloss FR, Kessler P, et al: Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clin Oral Implants Res 15:187, 2004 35. Fontana S, Olmedo DG, Linares JA, et al: Effect of platelet-rich plasma on the peri-implant bone response: An experimental study. Implant Dent 13:73, 2004 36. Fuerst G, Gruber R, Tangl S, et al: Enhanced bone-to-implant contact by platelet-released growth factors in mandibular cortical bone: A histomorphometric study in minipigs. Int J Oral Maxillofac Implants 18:685, 2003 37. Aghaloo TL, Moy PK, Freymiller EG: Investigation of plateletrich plasma in rabbit cranial defects: A study. J Oral Maxillofac Surg 60:1176, 2002 38. Choi BH, Im CJ, Huh JY, et al: Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 33:56, 2004 39. Jensen TB, Rahbek O, Overgaard S, et al: Platelet-rich plasma and fresh frozen bone allograft as enhancement of implant fixation. An experimental study in dogs. J Orthop Res 22:653, 2004 40. Butterfield KJ, Bennett, Gronowicz G, et al: Effect of plateletrich plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model. J Oral Maxillofac Surg 63:370, 2005 41. Jensen TB, Rahbek O, Overgaard S, et al: No effect of plateletrich plasma with frozen or processed bone allograft around noncemented implants. Int Orthop 29:67, 2005 42. 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 16:250, 2005 43. Sanchez AR, Sheridan PJ, Eckert SE, et al: Influence of plateletrich plasma added to xenogeneic bone grafts in periimplant defects: A vital fluorescence study in dogs. Clin Implant Dent Relat Res 7:61, 2005
Effects of Platelet-Rich Plasma at the Cellular Level on Healing of Autologous Bone-Grafted Mandibular Defects in Dogs David Gerard, PhD,* Eric R. Carlson, DMD, MD,† Jack E. Gotcher, DMD, PhD,‡ and Mykle Jacobs, DDS§ Purpose: This study describes the effect of platelet-rich plasma (PRP) at the cellular level on immediate
autologous bone grafts in dog mandibles. Materials and Methods: Twelve adult dogs weighing 40 to 50 pounds received bilateral inferior mandibular border resections measuring 2 cm ⫻ 1 cm. The right side was grafted with milled autologous iliac corticocancellous bone along with 2 cc of PRP taken from the same animal. The left side had the same amount of milled bone placed in the defect without PRP. Three animals were sacrificed at 1, 2, 3, and 6 months postsurgery. Ten and 3 days before sacrifice, all dogs received 10 mg/kg body weight of intravenous tetracycline. At sacrifice, the grafts along with adjacent native bone were harvested and immediately fixed in Carson’s fixative for 48 to 72 hours. The samples were then dehydrated over a 2-week period in a graded ethanol series and embedded in Spurr’s plastic. Two 100- sections from the center of each graft were cut, mounted on glass slides, ground to 40 , and stained. A digitally generated grid was superimposed over each section, to give 32 fields of 2.5 mm2. Each of these fields was examined at a magnification of ⫻100 to determine the number of osteoblasts and osteoclasts present. Results: The mean average of the total numbers of osteoblasts and osteoclasts were significantly higher in the PRP graft sites than in the non-PRP graft sites at 1 month. However, if specific fields were compared, then 14 of the 32 fields showed no difference in the number of osteoblasts and osteoclasts. At 2, 3, and 6 months, there was no significant difference in the total number of osteoblasts or osteoclasts in the PRP and non-PRP grafts, or in any of the 32 fields. Conclusions: At the cellular level, PRP increased the number of osteoblasts and osteoclasts recruited to the graft site at 1 month, and this overall increase was more evident at the superior and lateral margins of the graft than in other areas. Fields along the inferior margin showed the fewest number of cells for both the PRP and non-PRP grafts. At later times there was no significant difference in the number of osteoblasts and osteoclasts in the PRP and non-PRP graft sites in any region of the grafts. This study indicates that the increased number of osteoblasts and osteoclasts in the graft sites due to the addition of PRP was short-lived, and that autologous bone grafts without PRP had similar numbers of active bone cells after 1 month in this animal model. © 2007 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 65:721-727, 2007 The use of fresh, healthy autologous corticocancellous bone remains the gold standard for bone graft reconstruction of the jaws. Many other materials have been used to repair bony defects, but their efficacy is still measured by how they compare with autologous grafts.
Researchers have looked for ways to enhance xenograft materials by various means, including the addition of autologous bone,1 the addition of growth factors2-4 or the use of guided tissue regeneration membranes.5,6 However, if the patient is not compro-
Received from the Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of Medicine, Knoxville, TN. *Associate Professor, Director of Research. †Professor and Chairman. ‡Professor. §Formerly, Chief Resident; Currently, Fellow, Oral and Maxillofacial Surgery, St John’s Mercy Medical Center, St Louis, MO. Presented in part at the American Association of Oral and Max-
illofacial Surgeons’ 87th Annual Meeting and Scientific Sessions. Address correspondence and reprint requests to Dr Carlson: University of Tennessee Medical Center, Department of Oral and Maxillofacial Surgery, 1930 Alcoa Highway, Suite 335, Knoxville, TN 37920; e-mail: [email protected] © 2007 American Association of Oral and Maxillofacial Surgeons
0278-2391/07/6504-0020$32.00/0 doi:10.1016/j.joms.2006.09.025
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722 mised, then autologous bone grafts are still the most commonly used material for treating bony defects, especially of the craniofacial region. In recent years, researchers have looked for ways to enhance healing of autologous bone grafts by adding individual growth factors3 or PRP.7-12 The rationale behind using PRP is that activation of a large number of platelets at the time of graft placement releases a bolus of various growth factors,13 including platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, vascular endothelial growth factor (VEGF), insulinlike growth factor (IGF)-I, and epidermal growth factor (EGF).14 This increased concentration of growth factors is thought to have a positive effect on the recruitment of osteoblasts (ob) and osteoclasts (oc) precursors, differentiation and activation of osteoblasts and osteoclasts, and recruitment and activation of endothelial cells, enhancing angiogenesis. Autologous bone grafts heal through a mechanism whereby the body replaces the nonviable grafted bone with viable new bone. In a previous study, we reported on this process in a dog mandible bone graft model at the tissue level using histomorphometry.15 In that study, we described the effect of PRP on graft healing at 1, 2, 3, and 6 months postsurgery. We found that PRP increased the amount of new bone formed and the amount of nonviable grafted bone removed up to 2 months. After 2 months, there was no difference between the PRP and non-PRP graft sites. At the cellular level, many researchers, notably Frost,16 have studied osteoblast and osteoclast activity and morphology in normal bone. In addition, osteoblast and osteoclast activity and morphology have been described for some skeletal diseases, including Paget’s disease,16 osteoporosis,17 and osteopetrosis.18,19 To the best of our knowledge, there are no published studies examining bone graft healing at the cellular level. Because osteoblasts and osteoclasts are doing the work of bone formation and resorption, it is important to understand the effect of PRP on these cells. The hypothesis of this study is that the addition of PRP will increase the number of osteoblasts and osteoclasts in autologous mandibular bone grafts at 1 and 2 months, but not at 3 and 6 months. This hypothesis is based on the results of our previous study that showed increased formation of new bone and resorption of nonviable bone at the tissue level after 1 month and 2 months.15 Quantitative analysis of cell numbers was performed on stained sections. Our findings yield valuable information on the effect of PRP at the cellular level on autologous mandibular bone grafts. They show that the influence of PRP on early bone graft healing is localized to certain defined regions of the grafts in this animal model.
PLATELET-RICH PLASMA AND GRAFT HEALING
Materials and Methods PRP PREPARATION
PRP was prepared using a Harvest Technologies Smart Prep Centrifuge, with the PRP-20cc prep kit (Harvest Technologies, Plymouth, MA). A 20-cc sample of peripheral blood was drawn from each animal and processed for PRP preparation using the manufacturer’s instructions. An additional 2 cc of blood was drawn at the same time to calculate peripheral platelet counts for each animal. SURGICAL PROCEDURE
The surgical procedure for donor site bone harvest and recipient tissue site preparation and grafting was standardized and reported in a previous publication.15 TISSUE PROCESSING
Three animals were killed at 1, 2, 3, and 6 months postsurgery. After euthanasia, the mandibular defects and adjacent mandible (2 to 3 mm beyond the screws) were harvested intact and immediately placed into Carson’s fixative. Within the first 24 hours, standardized radiographs of the samples were obtained. After 48 hours of fixation, the undecalcified samples were dehydrated in an ethanol series over a 2-week period and embedded in Spurr’s plastic. Serial longitudinal 100- sections were cut through the graft sites and adjacent mandible with a Leitz 1600 bone saw (Leitz, Oberkochen, Germany). QUANTITATIVE CELL COUNT ANALYSIS
Two sections from the center of each defect were glued onto petrographic slides and ground to a thickness of 40 . The sections were stained with alizarine red, Sanderson’s rapid bone stain, and/or light green stain. The slides were examined at ⫻100, and a grid of 32 equal-sized fields of 2.5 mm2 was superimposed over the graft sites using Image-Pro Express software (Media Cybernetics, Silver Spring, MD) interfaced with a Leica DMRX research microscope (Leica Microsystems, Wetzlar, Germany) and a Sony digital camera (Sony, Tokyo, Japan) (Fig 1A). An image of each field was digitally captured. The area within each field was then examined at higher magnification (⫻250) to accurately identify and count all cells within the field. Thus 64 fields (32 from each section) were examined for each graft site, and 192 fields (64 from each of 3 animals) were examined for each time and treatment (PRP or non-PRP). The total number of osteoblasts (Fig 2) and osteoclasts (Fig 3) from each section, as well as from each field, was recorded.
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FIGURE 1. A, This schematic drawing depicts the 32-field grid overlay on a section of the 1 cm ⫻ 2 cm graft site. The fields are numbered sequentially from anterior to posterior and superior to inferior. Each field is 2.5 mm2. B, This figure shows the mean average of the number of osteoblasts (ob) and osteoclasts (oc) in each of the 32 fields analyzed at 1 month for both the PRP and non-PRP grafts. The numbers in red indicate a significant difference in the number of osteoblasts or osteoclasts counted in those fields. P ⬍ .05. Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
STATISTICAL ANALYSIS
The number of osteoblasts and osteoclasts counted for each section was calculated for each time and treatment (n ⫽ 6, 2 sections from each graft site and 3 animals each time). The mean average of the total number of osteoblasts and osteoclasts for PRP and non-PRP grafts were compared for each time using 1-way analysis of variance (ANOVA) (Figs 4, 5). Also, the mean averages of osteoblasts and osteoclasts for each of the corresponding 32 fields were compared using 1-way ANOVA. For example, the mean average of the number of osteoblasts in field 1 was compared for
the PRP and non-PRP grafts for each time (n ⫽ 6, field 1 from 2 sections and 3 animals for each time point and treatment). Differences in the number of osteoblasts or osteoclasts in the PRP and non-PRP grafts sites were considered significant at P ⬍ .05.
Results PRP PREPARATION
Platelet concentration averaged 388% for 1-month animals, 344% for 2-month animals, 386% for 3-month animals, and 334% for 6-month animals.15 Two ani-
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PLATELET-RICH PLASMA AND GRAFT HEALING
FIGURE 4. Comparison of the mean average number of osteoblasts in PRP and non-PRP grafts for 1, 2, 3, and 6 months. *Indicates a significant difference, P ⬍ .05. Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
FIGURE 2. This photograph shows numerous active osteoblasts on the forming surface of new bone. This section was from a non-PRP graft at 3 months stained with Sanderson’s rapid bone stain and light-green stain. (Original magnification ⫻250.) Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
mals at the 3-month time point did not have concentrations calculated. These platelet concentrations are comparable to those described by Marx and coworkers13,14 in their clinical trials. QUANTITATIVE CELL COUNT ANALYSIS
Comparing the mean average of the total number of osteoblasts found in each section of the PRP and non-PRP grafts revealed a significantly higher number
in the PRP grafts at 1 month (P ⬍ .05). This was also true for osteoclasts (P ⬍ .05). However, comparing the total number of osteoblasts and osteoclasts in the grafts at 2, 3, and 6 months revealed no significant difference (Figs 4, 5). To further refine possible differences in cellular activity in different areas of the bone graft, comparisons were made between each field and compared for the PRP and non-PRP grafts. Thus, all numbered fields (eg, the fields marked “1,” located at the most anterior/superior region of the graft [Fig 1]), were compared. By analyzing and comparing only fields in the same location in the graft sites, a clearer picture of where PRP may most influence graft healing emerged. The results of this analysis are shown for the 1-month time point in Figure 1B. In 14 of the 32 fields that cover the graft site, there was no significant difference in the number of osteoblasts and osteoclasts between the PRP and non-PRP graft sites; in the other 18 fields, the difference in the number of osteoblasts and osteoclasts was significant. Generally, the fields along
FIGURE 3. This photograph shows 2 osteoclasts on the surface of a Howship’s lacunae at 3 months in a PRP graft. The section was stained with Sanderson’s rapid bone stain and light-green stain. (Original magnification ⫻250.)
FIGURE 5. This shows the mean average number of osteoclasts in PRP and non-PRP grafts for 1, 2, 3, and 6 months. *Indicates a significant difference, P ⬍ .05.
Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
Gerard et al. Platelet-Rich Plasma and Graft Healing. J Oral Maxillofac Surg 2007.
GERARD ET AL
the anterior, posterior, and superior margins showed the greatest increase in the numbers of osteoblasts and osteoclasts when PRP was added to the graft. The effect of PRP was not significant along the inferior margin and toward the center of the graft site. Moreover, fewer osteoblasts and osteoclasts were counted along the inferior margin in both the PRP and non-PRP grafts; in some of these fields, very few osteoblasts and no osteoclasts were seen (Fig 1). At 2, 3, and 6 months, there was no statistically significant difference in the number of osteoblasts and osteoclasts with or without PRP in any of the 32 fields (Figs 4, 5).
Discussion This is the first study to report on the effect of PRP on autologous bone grafts at the cellular level. Using digital image capture techniques, it was possible to subdivide each graft section into 32 equal fields of 2.5 mm2 on slides of 2 central longitudinal sections of each graft. In this way, all osteoblasts and osteoclasts within each field were counted. Comparing the total number of osteoblasts and osteoclasts from all fields for the PRP and non-PRP grafts showed that the PRP grafts had significantly more osteoblasts and osteoclasts at 1 month, but there was no significant difference at 2, 3, or 6 months. Because the 32 fields were reproduced over each section, it was also possible to examine cell numbers for each field separate from the whole graft. In doing so, results were further refined to demonstrate that even at 1 month, not all areas of the graft showed a difference in the numbers of osteoblasts and osteoclasts. Thus, fields at the anterior and posterior margin and at the superior edge of the graft were most significantly affected by the addition of PRP. The fields at the center of the graft and at the inferior margin generally showed no difference in cell numbers with the addition of PRP. Factors that may contribute to an increased effect of PRP on the anterior and posterior regions of the graft include early angiogenesis in these regions due to high concentrations of EGF and PDGF that stimulate endothelial cell proliferation. In addition, increased concentrations of TGF- and IGF-I stimulate both osteoblast and osteoclast precursor cells to divide and differentiate. In the superior region of the graft, these growth factors, along with the presence of a robust neurovascular bundle in this animal model, probably stimulate early angiogenesis as well as osteoblast and osteoclast activity. The Utah paradigm stresses the important role of mechanical forces on bone healing.20 Removal of a significant portion of a weight-bearing bone will have a significant effect on bone activity by increasing the load on the areas adjacent to the graft. This effect will most likely be seen along the margins of the defect that are in con-
725 tact with native bone placed under an increased load. Although every effort was made to maintain the periosteum, its disruption during surgical placement of the grafts may have played a role in PRP’s lack of significant effect on the numbers of osteoblasts and osteoclasts along the inferior border of the graft. In addition, consolidation of the grafts in this region may have caused loss of bone from these areas. This possibility is supported by the fact that few osteoblasts or osteoclasts were found in many fields in this area in either the PRP or non-PRP grafts at 1 month. Osteoclast size and numbers of nuclei have been reported to be significant factors in determining the activity of these cells.21,22 In the present study, we found no observable difference in the size of osteoclasts or in the number of nuclei in these cells between the PRP and non-PRP grafts. This finding suggests that PRP did not affect the activity of the cells but only increased the number of osteoclasts at 1 month. In our earlier study,15 we reported a significant increase in new bone in the PRP grafts at 2 months, whereas in the present study we found no significant difference in the number of osteoblasts at 2 months. This difference can be explained by the fact that the higher number of osteoblasts at 1 month would contribute to greater new bone formation. This increase would be seen even at 2 months at the tissue level. Some studies have reported long-term positive effects of PRP on bone grafts. Marx et al13 first described the effect of PRP on mandibular bone grafts in a series of 84 patients. These authors reported that the addition of PRP significantly enhanced autologous bone grafts out to 6 months over non-PRP grafts. Wiltfang et al23 reported on 39 patients receiving sinus floor augmentation with either beta-tricalciumphosphate (beta-TCP) alone or with PRP. At 6 months postsurgery, the PRP-added grafts had 8% to 10% more new bone, but the PRP did not increase the rate at which the beta-TCP was resorbed. Kassolis and Reynolds24 reported on 10 patients who received bilateral maxillary subantral sinus augmentation, with 1 site receiving freeze-dried bone allograft (FDBA) plus PRP and the other side receiving FDBA with a resorbable membrane. At 4.5 and 6 months postsurgery, the PRP-treated grafts showed a higher percentage of vital tissue and bone formation and a smaller amount of FDBA, suggesting that the PRP enhanced both bone formation and the removal of FDBA. Lekovic et al25 reported on 52 grade II mandibular molar furcation defects treated with either open flap debridement or bovine porous bone mineral (BPBM) along with PRP and guided tissue regeneration (GTR) membrane. At 6 months posttreatment, the sites were evaluated clinically, and the BPBM/PRP/GTR sites showed better results. These authors stated the need
726 for further studies to determine what role each of the 3 components of the experimental treatment played in the improved results. Mazor et al26 reported on 105 patients who received sinus augmentation with a composite graft of 30% to 40% autogenous bone and 60% to 70% xenograft, along with PRP. Dental implants were placed at the time of augmentation and uncovered at 6 months, at which time all implants were osseointegrated. There were no controls for this study. A growing body of literature showing that PRP enhances graft healing early, but not long-term, supports our findings in the present study. Those earlier studies investigated the use of PRP in a number of different applications in conjunction with autologous bone grafts or with various allograft materials. For example, Hanna et al27 examined bovine-derived xenografts (BDX) with and without PRP in 13 patients with bilateral periodontal defects. At 6 months, there was no difference between the PRP and non-PRP sites with reference to a number of clinical measurements. No earlier time points were used for this study. Lekovic et al28 examined the used of BPMB either with PRP or with PRP plus GTR in 21 patients with bilateral periodontal defects and measured various clinical parameters at 6 months posttreatment. Both treatments showed significant improvement, but there was no significant difference between the 2 groups. A number of animal studies have investigated the efficacy of PRP in conjunction with various grafting materials at various times. Schiegel et al29 used a pig model to study the effect of PRP on autogenous grafts or a bovine collagen device using both microradiography and light microscopy. They found that PRP had a significant positive effect on autogenous grafts at 2 weeks, but did not enhance the bovine collagen device. At 12 weeks, there was no difference between the PRP and non-PRP treatment groups. Kovacs et al30 investigated the effect of PRP combined with betaTCP on mandibular defects in dogs using densitometric and histological techniques and found that PRP enhanced bone formation at 6 and 12 weeks. No later time points were examined in this study. Suba et al31 examined the healing of premolar extraction sockets in dogs when Cerasorb or Cerasorb with PRP were placed in the sockets. At 6 and 12 weeks, more new bone was found in the PRP sites, but at 24 weeks, there was no difference between the PRP and nonPRP sites. Grageda et al32 compared the use of 2 allograft materials with and without PRP in sinus augmentation in sheep. Histomorphometric analysis indicated that PRP did not enhance bone formation at either 3 or 6 months. No earlier time points were studied. Numerous other studies have examined the effect of PRP on different bony defects in animal models.
PLATELET-RICH PLASMA AND GRAFT HEALING
Some reported that PRP is effective in enhancing bone healing only early in the process,33-36 whereas others reported that PRP has no beneficial effect at any time studied.37-43 The present study further defines the role of PRP in the early enhancement of autologous bone grafts in the canine mandible. It appears that PRP enhances the number of osteoblasts and osteoclasts in the graft site only at 1 month. At 2, 3, and 6 months, no significant difference in the total number of osteoblasts and osteoclasts was seen in the PRP and nonPRP graft sites. Using digital imaging analysis of reproducible sites in each graft, we further determined that the enhancement of PRP was restricted to peripheral areas of the graft site that were in contact with native bone, and that this enhancement did not persist beyond 1 month. The results of the present study support a growing body of literature indicating that the positive effects of PRP on bone grafts are seen only early in the healing process.
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GERARD ET AL 13. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:48, 1998 14. Marx RE: Platelet-rich plasma: Evidence to support its use. J Oral Maxillofac Surg 47:489, 2004 15. Gerard D, Carlson ER, Gotcher JE, et al: Effects of platelet-rich plasma on the healing of autologous bone grafted mandibular defects in dogs. J Oral Maxillofac Surg 64:443, 2006 16. Frost HM: Intermediary Organization of the Skeleton. Boca Raton, FL, CRC Press, 1986 17. Reddy SV, Menaa C, Singer FR, et al: Cell biology of Paget’s disease. J Bone Miner Res 14(Suppl 2):3, 1999 18. Gruber HE, Ivey JL, Thompson ER, et al: Osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis. Miner Electrolyte Metab 12:246, 1986 19. Henriksen K, Gram J, Schaller S, et al: Characterization of osteoclasts from patients harboring a G215R mutation in CIC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 164:1537, 2004 20. Marks SC Jr: Osteoclast biology: Lesions from mammalian mutations. Am J Med Genet 34:43, 1989 21. Frost HM: A 2003 update of bone physiology and Wolff’s law for clinicians. Angle Orthod 74:3, 2004 22. Lees RL, Heersche JN: Macrophage colony-stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size. J Bone Miner Res 14:937, 1999 23. Wiltfang J, Schlegel KA, Schultze-Mosgau S, et al: Sinus floor augmentation with beta-tricalciumphosphate (beta-TCP): Does platelet-rich plasma promote its osseous integration and degradation? Clin Oral Implants Res 14:213, 2003 24. Kassolis JD, Reynolds MA: Evaluation of the adjunctive benefits of platelet-rich plasma in subantral sinus augmentation. J Craniofac Surg 16:280, 2005 25. Lekovic V, Camargo PM, Weinlaender M, et al: Effectiveness of a combination of platelet-rich plasma, bovine porous bone mineral and guided tissue regeneration in the treatment of mandibular grade II molar furcations in humans. J Clin Periodontol 30:746, 2003 26. Mazor Z, Peleg M, Garg AK, et al: Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: Patient series study. Implant Dent 13:65, 2004 27. Hanna R, Trejo PM, Weltman RL: Treatment of intrabony defects with bovine-derived xenograft alone and in combination with platelet-rich plasma: A randomized clinical trial. J Periodontol 75:1668, 2004 28. Lekovic V, Camargo PM, Weinlaender M, et al: Comparison of platelet-rich plasma, bovine porous bone mineral, and guided tissue regeneration versus platelet-rich plasma and bovine porous bone mineral in the treatment of intrabony defects: A reentry study. J Periodontol 73:198, 2002
727 29. Schiegel KA, Donnath K, Rupprecht S, et al: De novo bone formation using bovine collagen and platelet-rich plasma. Biomaterials 25:5387, 2004 30. Kovacs K, Velich N, Huszar T, et al: Histomorphometric and densitometric evaluation of the effects of platelet-rich plasma on the remodeling of beta-tricalcium phosphate in beagle dogs. J Craniofac Surg 16:150, 2005 31. Suba Z, Takacs, Gyulai-Gaal S, et al: Facilitation of beta-tricalcium phosphate-induced alveolar bone regeneration by platelet-rich plasma in beagle dogs: A histologic and histomorphometric study. Int J Oral Maxillofac Implants 19:832, 2004 32. Grageda E, Lozada JL, Boyne PJ, et al: Bone formation in the maxillary sinus by using platelet-rich plasma: An experimental study in sheep. J Oral Implantol 31:2, 2005 33. Jakse N, Tangl S, Gilli R, et al: Influence of PRP on autogenous sinus grafts: An experimental study on sheep. Clin Oral Implants Res 14:578, 2003 34. Wiltfang J, Kloss FR, Kessler P, et al: Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clin Oral Implants Res 15:187, 2004 35. Fontana S, Olmedo DG, Linares JA, et al: Effect of platelet-rich plasma on the peri-implant bone response: An experimental study. Implant Dent 13:73, 2004 36. Fuerst G, Gruber R, Tangl S, et al: Enhanced bone-to-implant contact by platelet-released growth factors in mandibular cortical bone: A histomorphometric study in minipigs. Int J Oral Maxillofac Implants 18:685, 2003 37. Aghaloo TL, Moy PK, Freymiller EG: Investigation of plateletrich plasma in rabbit cranial defects: A study. J Oral Maxillofac Surg 60:1176, 2002 38. Choi BH, Im CJ, Huh JY, et al: Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 33:56, 2004 39. Jensen TB, Rahbek O, Overgaard S, et al: Platelet-rich plasma and fresh frozen bone allograft as enhancement of implant fixation. An experimental study in dogs. J Orthop Res 22:653, 2004 40. Butterfield KJ, Bennett, Gronowicz G, et al: Effect of plateletrich plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model. J Oral Maxillofac Surg 63:370, 2005 41. Jensen TB, Rahbek O, Overgaard S, et al: No effect of plateletrich plasma with frozen or processed bone allograft around noncemented implants. Int Orthop 29:67, 2005 42. 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 16:250, 2005 43. Sanchez AR, Sheridan PJ, Eckert SE, et al: Influence of plateletrich plasma added to xenogeneic bone grafts in periimplant defects: A vital fluorescence study in dogs. Clin Implant Dent Relat Res 7:61, 2005