Int. J. Oral Maxillofac. Surg. 2007; 36: 132136 doi:10.1016/j.ijom.2006.06.004, available online at http://www.sciencedirect.

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Leading Research Paper Tissue Enhancement

Platelet-rich plasma does not improve bone regeneration around peri-implant bone defectsA pilot study in dogs
M. Z. Casati, B. C. de Vasconcelos Gurgel, P. F. Gonc alves, S. P. Pimentel, G. da Rocha Nogueira Filho, F. H. NocitiJr. E. A. Sallum: Platelet-rich plasma does not improve bone regeneration around peri-implant bone defectsA pilot study in dogs. Int. J. Oral Maxillofac. Surg. 2007; 36: 132136. # 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The purpose of the present study was to evaluate the inuence of plateletrich plasma (PRP) on bone regeneration in dehiscence-type bone defects around dental implants. Ten male adult mongrel dogs were used. Three months after teeth extractions, an osteotomie for implantation and a buccal dehiscence defect were prepared on both sides of the jaws. Two dental implants with machined surfaces were placed on each implant site of the mandible. Dehiscences were randomly assigned to the following groups: (1) test (PRP) and (2) control. After 3 months animals were sacriced; implants and adjacent hard tissues were processed for undecalcied sections. Bone-to-implant contact (BIC), bone density (BD) within the limits of implant threads, bone density (BO) and new bone area (NB) in a zone lateral to the implant, corresponding to bone defects, were obtained and measured. Inter group analysis (paired Students t-test, a = 5%) demonstrated no statistically signicant differences for any of the parameters when PRP was used (P > 0.05). Within the limits of the present study, it was concluded that platelet-rich plasma alone did not enhance bone regeneration for peri-implant defects.

M. Z. Casati, B. C. de Vasconcelos Gurgel, P. F. Gonc alves, S. P. Pimentel, G. da Rocha Nogueira Filho, F. H. Nociti Jr., E. A. Sallum
Department of Prosthodontics and Periodontics, Division of Periodontics, School o of Dentistry at Piracicaba/Unicamp, Sa Paulo, Brazil

Key words: platelet-rich plasma; growth factors; bone regeneration; dental implants; animal study. Accepted for publication 20 June 2006 Available online 4 August 2006

New techniques have been researched that will promote or enhance bone healing in maxillofacial surgery and implantology17; the use of platelet-rich plasma (PRP) is an example of such a technique. Its use is based on the potential of the plasma to release multiple wound-healing growth factors and cytokines16,23, which are responsible for increasing cell mitosis,
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increasing collagen production, recruiting other cells to the site of injury, initiating vascular in-growth and inducing cell differentiation5. Previous studies have demonstrated favourable clinical results with PRP application in oral surgery1,16,23. Released growth factors such as platelet-derived growth factor (PDGF), transforming

growth factor b (TGF b), insulin-like growth factor (IGF) and epidermal growth factor (EGF) have been shown to have an osteoregenerative effect on peri-implant bone15,21 because of their pro-angiogenic effects and differentiating effects on osteoblasts1. The use of an autologous source of these growth factors, i.e. platelet-rich plasma (PRP), released from

# 2006 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Platelet-rich plasma does not improve bone regeneration around peri-implant bone defects
platelet activation, could provide increased bone regeneration around dental implants when associated or not with bone grafts11,12,24. Some animal studies have analysed the osseointegration rate and observed that PRP application showed higher values of bone-to-implant contact as well as bone density6,24. On the basis of early positive results for PRP utilization, the purpose of the present study was to evaluate the effect of platelet-rich plasma on bone regeneration at peri-implant dehiscence-type defects.
Materials and methods

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rejected from each tube and the remaining content was centrifuged again at 1200 r.p.m. for 15 min. The middle portion corresponding to the platelet-rich plasma was pipetted from each tube and 10% calcium chloride was added to the preparation to activate platelets. The platelet gel was formed after 10 to 15 min. A measured increase of 320% was observed in the platelet concentrate. A mean value of peripheral blood platelet counts yielded 148,520 platelets/ml and the platelet count of PRP was 460,350 platelets/ml.
Defect treatment

sutures. In the dehiscences of the control group, after implant installation, the mucoperiosteal ap was immediately repositioned and sutured. After surgery, 1 mg/kg of banamine was administered by subcutaneous injection for 3 consecutive days, 20,000 i.u. of penicillin/erythromycin was administered in one dose and after 4 days. Fifteen days postsurgically, the sutures were removed. For 1 month, postsurgery, oral hygiene was performed with 0.12% chlorhexidine gluconate spray daily and prophylaxis was also carried out once a month until the end of the study.
Histology and histomorphometry

This study was initially approved by the Institutional Committee for Ethics in Animal Research of the University of Campinas. Ten male adult mongrel dogs, 25 kg in weight, were used in the present study. General anaesthesia was obtained by intravenous injection of 25% sodium thiopental solution (1 ml/kg) supplemented with local administration of 2% lidocaine (1:100,000). After full thickness aps elevation, the second, third and fourth mandibular-premolars (P2, P3 and P4) were extracted bilaterally. The extraction sites were allowed to heal for 3 months prior to implant placement. Mucoperiosteal aps were raised and two implant osteotomies were prepared in the posterior region, one on each side of the edentulous areas of the mandible. Before implant placement, one dehiscence-type defect (4 mm 5 mm) was bilaterally created on the buccal aspect of the implant osteotomies with a high rotation conic bur with sterile continuous saline solution irrigation. Two 4 mm 8.5 mm machined screw-shaped commercial pure titanium implants were placed on each side of the mandible, and the soft tissues were repositioned and sutured.
PRP preparation

After implant placement, the dehiscence defects were randomly assigned for treatment according to the following groups: (1) test (PRP) and (2) control. Before suturing, the PRP gel was applied on the dehiscence defects of the test group and the mucoperiosteal aps were then repositioned and sutured with nonresorbable

At 3 months postsurgery, the animals were sacriced and the mandibles were removed and xed in buffered 10% formalin solution. Block sections were made from the implants and undecalcied sections were prepared as previously described by DONATH & BREUNER4. One

Blood was obtained several minutes before starting surgery, prior to the administration of anaesthesia. Three 5-ml tubes containing 0.5 ml of 3.2% sodium citrate solution as an anticoagulant were drawn from each dog. The tubes were centrifuged at 1200 r.p.m. for 10 min at room temperature. The blood was then separated into three following parts: red blood cells (at the bottom of the tube), platelet-rich plasma (a discrete grey line in the middle of the tube) and platelet-poor plasma (at the top of the tube). The portion corresponding to the platelet-poor plasma was

Fig. 1. Schematic drawing illustrating the histometric parameters evaluated. Buccal-lingual view of the implant showing new bone formation outside the threads (nbo) and within the threads (nbw). Coloured lines exhibit new bone-to-implant contact (blue), new bone density within the threads (red) and new bone density and area outside the threads (green).

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slide was created for each implant and the sections were stained with 1% toluidine blue. The percentage of bone-to-implant contact and bone density within the region above the reversion bone line in the defect area, as well as bone density and bone area outside the threads, were measured using an image analysis system (Image Pro1, Media Cybernetics, Silver Spring, MD, USA), considering the bone tissue formed above the reversion bone line (Fig. 1).
Statistical analysis

Fig. 3. Mean percentage and standard deviations of bone density within threads (BD) obtained after treatment of peri-implant bony defects in group test (PRP) and control (no PRP). Paired Student t-test, d.f. = 9, P = 0.8638.

The data were analysed using the paired Students t-test to test the hypothesis that there were no differences in the parameters at the defect areas for all groups. Values of P < 0.05 were considered statistically signicant.
Results

Fig. 4. Mean percentage and standard deviations of bone area out of the threads in mm2 (NB) obtained after treatment of peri-implant bony defects in group test (PRP) and control (no PRP). Paired Student t-test, d.f. = 9, P = 1.

All 20 submerged dental implants healed with no complications. Signicant differences were not observed in any of the parameters for the use of PRP. Within threads, the percentage of bone-to-implant contact (group 1: 9.82 13.68% vs. group 2: 9.02 18.93%) and bone density (group 1: 34.05 23.29% vs. group 2: 32.48 28.81%) did not present any signicant difference when PRP was used (Figs 2 and 3, respectively). Similarly, for the out of threads parameter, no signicant differences were observed for the bone area (group 1: 1.31 2.28 mm2 vs. group 2: 1.32 1.17 mm2) (Fig. 4) and the bone density (group 1: 84.81 13.90% vs. group 2: 88.89 10.22%) (Fig. 5) when PRP was applied. The histometric results are illustrated in Fig. 6.
Discussion

Fig. 5. Mean percentage and standard deviations of bone density out of the threads (BO) obtained after treatment of peri-implant bony defects in group test (PRP) and control (no PRP). Paired Student t-test, d.f. = 9, P = 0.6253.

The present study evaluated bone regeneration at dehiscence-type defects using platelet-rich plasma at different implant surfaces. Dehiscence- and fenestrationtype bone defects are frequently associated with dental implants and can com-

promise their use18. Traditionally, these defects could be treated by guided bone regeneration (GBR), associated or not with bone grafts to promote higher bone volume8, depending on space maintenance promoted with membrane application. Growth factors have been previously tested around dental implants and have been demonstrated to have an osteoregenerative effect on peri-implant bone15,21 because of their pro-angiogenic effects and differentiating effects on osteoblasts1. Thus, the present study aimed to analyse

Fig. 2. Mean percentage and standard deviations of bone-to-implant contact (BIC) obtained after treatment of peri-implant bony defects in group test (PRP) and control (no PRP). Paired Students t-test, d.f. = 9, P = 0.9157.

the inuence of platelet-rich plasma, an autologous source of growth factors, on the bone regeneration of peri-implant defects. In all surgically created bone defects treated, bone regeneration could be observed without differences between groups, although it did not completely ll the defects. Platelet-rich plasma application was initially proposed to enhance wound healing16,23. The platelet concentrate is formed by mixing PRP, derived from autologous whole blood, with thrombin and/or calcium chloride20. The biological theory surrounding the use of PRP is based on the presence of growth factors in the platelet a-granules, such as platelet-derived growth factor, transforming growth factor b , insulin-like growth factor 1, epidermal growth factors, all of which are released after platelet activation1,15. These growth factors are considered as critical for the regulation and stimulation of wound healing, regulating cellular processes such as mitogenesis, chemotaxis, differentiation and metabolism7. Although

Platelet-rich plasma does not improve bone regeneration around peri-implant bone defects

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tions of PRP, associated or not, with other well-known regenerative therapies, such as guided bone regeneration and bone grafts, could be necessary to elucidate the real impact of PRP on bone regeneration around dental implants. Thus, within the limits of the present study, it may be concluded that the effects of platelet concentrates used alone are not sufcient to obtain bone regeneration around dental implants.
Acknowledgements. This study was sup o de Amparo a ported by Fundac a ` Pesquisa o Paulo FAPESP, prodo Estado de Sa cess 02/13387-6. We would also like to thank the histological technician, Mariana Piovezzan Fuggolin, who contributed to this study with the preparation of slides.

Fig. 6. Photomicrographs illustrating the histological ndings for each group (A, PRP and B, control) in the previously exposed threads. The surgically produced dehiscence defect was not completely lled with new bone. Limited new bone (nb) formation could be observed, in both groups, only in the apical portion of the defect and was stained by a darker blue (original magnication 6.25, undecalcied sections, toluidine blue staining, bar = 1 mm).

previous studies have demonstrated the enhancement of the early cascade of tissue repair15,21, the platelets from platelet-rich plasma also showed large numbers of native growth factors22. The present study showed that PRP applied alone at bone defects did not have any additional effect on bone regeneration with regard to the parameters evaluated, such as bone-to-implant contact, bone area and bone density. According to KOVE13 KER , the effects of growth factor could be lost even before the osteogenic process occurs. More recent studies, such as that by JENSEN et al.9, have also not found any increased bone regeneration with PRP and bone grafts association in surrounded periimplant bone defects, even with high platelet concentrations (1,884,000 platelets/ ml). In addition, SANCHEZ et al.19 observed that PRP and bone grafts association did not alter bone mineral density or graft maturity levels. JENSEN et al.9 suggest that indications, a minimum level of platelets and the benet of using commercialized products need to be elucidated. The platelet concentration may represent an explanation for the results of the present study. A platelet concentration of 320% was observed (mean of 460,000 platelets/ml). It is possible that this concentration was not sufcient to promote bone formation. It has been reported that 1,000,000 platelets/ml may be necessary to achieve biological benets for wound healing. Outcomes from some studies have demonstrated increased bone formation with this concentration11,16,24. Below this range the effect is suboptimal; beyond this range, there may be a paradoxically inhibitory effect. However, WEIBRICH et al.22 did not observe differences in

bone-to-implant contact using various platelet concentrations (163,000 to 373,000; 503,000 to 1,729,000 and 1,845,000 to 3,200,000 platelets/ml). There has been no discussion regarding the ideal concentrations of platelet and growth factors to promote bone regeneration10. To date, there is insufcient information about growth factor interactions and how they inuence the activations of gene expression and protein production. LACOSTE et al.14 reported that growth factors may act at specic times and at appropriate concentrations and this may be another explanation for the absence of positive effects with PRP. As well as platelet concentrations, cell competition between nonosteogenic and osteogenic cells at the site may explain the absence of difference between the groups for the bone-to-implant contact data and the out of threads parameters2. In addition, soft tissue pressure could have led to a collapse and consequent reduction in bone regeneration, where bone healing occurred only in the apical portion of the defect. It has also been reported that the periosteum alone is not capable of generating new bone around exposed implants3. Although some positive effects have been observed to enhance bone regeneration in previous studies6,24, most papers which advocate the use of PRP compare platelet-rich plasma with an inappropriate control. Further studies which intend to demonstrate the benets of PRP should use a suitable control, i.e. platelet-poor plasma (PPP) or platelet-rich brin (PRF). According to the present study, PRP applied alone did not present any benets to bone healing. Therefore, additional information using higher concentra-

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