Injury, Int. J. Care Injured 41 (2010) 717–723

Combined use of platelet-rich plasma and autologous bone grafts in the treatmentof long bone defects in mini-pigs

M. Hakimi a, P. Jungbluth a,*, M. Sager b, M. Betsch a, M. Herten c, J. Becker c, J. Windolf a, M. Wild a

a Heinrich Heine University Hospital Duesseldorf, Department of Trauma and Handsurgery, Moorenstr. 5, 40225 Duesseldorf, Germanyb Heinrich Heine University Hospital Duesseldorf, Animal Research Institute, Duesseldorf, Germanyc Heinrich Heine University Hospital Duesseldorf, Department of Oral Surgery, Duesseldorf, Germany

A R T I C L E I N F O

Article history:

Accepted 8 December 2009

Keywords:

Platelet-rich plasma

Bone healing

Bone defect

Growth factors

Animal model

Mini-pig

A B S T R A C T

The use of platelet-rich plasma (PRP) for improving of bone defect healing is discussed controversially.

The aim of this study was to assess the effect of PRP in combination with autologous cancellous graft on

bone defect healing in a critical metaphyseal long bone defect. A critical size defect in the tibial

metaphysis of 16 mini-pigs was filled either with autologous cancellous graft as control group or with

autologous cancellous graft combined with autologous PRP. Compared to native blood platelets were

enriched about 4.9-fold in the PRP. After 6 weeks, the specimens were assessed by X-ray and histological

evaluation. Histomorphometrical analysis revealed that the area of new bone was significantly higher in

the PRP group concerning the central area of the defect zone (p < 0.02) as well as the cortical defect zone

(p < 0.01). All defects showed substantial new bone formation, but only defects of the PRP group

regenerated entirely. The PRP group was superior to the control group even in the semi-quantitative

assessment of the osseous bridging in both observed areas of the defect. Within the limits of the present

study it could be demonstrated that PRP combined with autologous cancellous graft leads to a

significantly better bone regeneration compared to isolated application of autologous cancellous graft in

an in vivo critical size defect on load-bearing long bones of mini-pigs.

� 2009 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

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Introduction

Despite the variety of implants and their further development,which enables a fracture stabilisation with the least possibleimpairment of soft tissues, about 10% of all fractures result in adelayed union.35 Patients with risk factors have even an increasedrisk to about 30%.35 The autologous cancellous graft in combinationwith suitable stable implants is considered the gold standard amongpseudoarthrosis.18 As long as this process does not result in thehealing of a fracture, alternative procedures must be taken. The useof osteoconductive and osteoinductive bone substitutes are suchalternatives or supplementary therapies.13 Nevertheless, with aconsiderable number of patients a refractory pseudoarthrosiscannot be avoided. Although the pathophysiology of fracturehealing has been the object of intensive research during the pastyears and many local mediators and their role in fracture healinghave been clarified, our knowledge has not yet enabled us to avoidthe appearance of non-unions.21,26 The role of the growth factors,their localisation, their specific characteristics and their applicationare discussed controversially.2,5,6,20,24 Thus the role of bone

* Corresponding author. Tel.: +49 211 8104410; fax: +49 211 8104902.

E-mail address: pascal.jungbluth@gmx.de (P. Jungbluth).

0020–1383/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.injury.2009.12.005

morphogenetic proteins (BMPs), which belong to the super familyof the transforming growth factor b (TGFb) have been reported onseveral occasions.9,14 Also, the proof of the release of such growthfactors as platelet-derived growth factor (PDGF), TGFb, vascularendothelial growth factor (VEGF), epidermal growth factor (EGF),insulin-like growth factor 1 (IGF-1) and platelet factor 4 (PF-4) fromplatelets has been reported.20,31,34 Thereby PDGF stimulatesmitogenesis of the marrow stem cells and endosteal osteoblaststransferred in the graft to increase their numbers by several orders ofmagnitude. TGFb activates the fibroblasts and preosteoblasts tomitose and increase their numbers and it supports their differentia-tion toward mature functioning osteoblasts.20,21 It also inhibits theformation of osteoclasts and bone resorption.23

Using platelet-rich plasma (PRP), which is produced from thepatient’s own blood, with a distinctly higher concentration ofplatelets in comparison to native blood, positive effects of thesegrowth factors could be shown.5,6,12,16 The existence of receptorsfor these growth factors were also found in the cells of theautologous cancellous graft.20 The knowledge about the existinggrowth factors in PRP and their osteoinductive characteristics, aswell as the corresponding receptors for these growth factors in theautologous cancellous graft allows the combined use of PRP andthe autologous cancellous graft as a possible successful method topromote bone healing.

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723718

The object of this study was to show the possible superiority ofa combined use of PRP and autologous cancellous graft comparedto the use of solely autologous cancellous graft for the treatment ofclinically relevant, so-called ‘‘critical size defects’’ of the load-bearing bone. A defined defect model of the proximal tibia of min-pigs was used in this study. Animal selection, management andsurgery protocol were approved by the local Animal Care and UseCommittee.

Material and methods

Animals

Due to preliminary case calculations sixteen female Goettingermini-pigs (aged 18–30 months, weight 25–35 kg) were used for anadequate statistical power in this study. The experimental segmentof the study started after an adaptation period of 2 weeks.

Study design and surgical procedure

The animals were randomised into the PRP and the controlgroup. The process of randomisation was according to the twodeliveries of eight mini-pigs each. Every group comprised of eightmini-pigs. The first delivery was treated without PRP, whereas thesecond delivery additionally received PRP. According to Wheeleret al.33 in all animals an 11 mm cylindrical defect from medial tolateral to a depth of 25 mm at the right proximal tibia withoutlateral cortical penetration was surgically created using acannulated reamer (Aesculap AG & Co. KG, Tuttlingen, Germany).In one group the defect was filled with autologous cancellous graftand in the other one with autologous cancellous graft incombination with PRP. In order to avoid a negative influence onbone healing the surgical approach, the size of the incision and thehandling of soft tissues in the defect area were identical in bothgroups. The duration of the surgery was identical in both groups,due to simultaneous preparation of the PRP by a co-worker. In allcases the animals were permitted to walk under full weightbearing immediately after surgery. None of them suffered from atibial fracture or had to be given up ahead of schedule.

All animals were starved for a minimum of 12 h before surgery.Antibiotic preparation was given once to each animal in aperioperative way as single shot of 3.3 ml lincomycin (lincomycin20%, WDT, Garbsen, Germany). After intramuscular sedation with0.5 mg/kg atropin (Atropinsulfat, B Braun, Melsungen, Germany),5 mg/kg azaperon (Stresnil1, Janssen-Cilag GmbH, Neuss, Ger-many) and 10 mg/kg ketamin (Ketavet1, Pharmacia GmbH,Karlsruhe, Germany), anaesthesia was initiated using 0.5 gthiopental (Inresa Arzneimittel GmbH, Freiburg, Germany). Forall surgical procedures, inhalation anaesthesia was performed byuse of oxygen and nitrous oxide and isoflurane. To maintainhydration and cardiac protection, all animals received a constantrate infusion of 5% glucose’s solution (Delta-Select, Pfullingen,Germany) combined with 10 ml Inzolen (Koehler Chemie GmbH,Alsbach-Hahnlein, Germany) and 5 ml lidocain 2% (lidocain-HCl, B.Braun, Melsungen, Germany) while anaesthetised. Intraoperativeanalgesia was performed by intravenous injection of 0.4 mg/kgPiritramid (Dipidolor1, Janssen-Cilag GmbH, Neuss, Germany) and4.5 mg/kg Carprofene (Rimadyl1, Pfitzer Pharma GmbH, Karlsruhe,Germany). For postoperative treatment, Piritramid and Carprofenewere applied subcutaneously for 3 days at the same dose.Additionally, prophylactic administration of Lincomycin (3,3 mllincomycin 20%, WDT, Garbsen, Germany) was performed post-operatively for 3 days.

All surgical procedures took place under aseptic conditions andwere performed by the same experienced surgeon. Prior to surgery,in the PRP group, 120 ml of whole blood was retrieved from the

jugular vein of the mini-pig. The right limb, distal to mid-thigh, andleft iliac crest were shaved, disinfected by Cutasept1 (Bode,Hamburg, Germany) and sterilely draped. An incision was madeover the left iliac crest and sharp dissection was used to expose thebone. A Kirschner wire (K-wire) was inserted in the iliac crest. Thecancellous graft was harvested from the iliac crest using the samecannulated reamer of 11-mm calibre. The size of this harvestedgraft was almost identical to the proximal tibial defect we createdbefore. In all cases the amount of cancellous graft was adequate tofill the defect.

The donor site was packed with gauze sponges until bleedingwas controlled and the incision closed in layers. The right proximaltibia was exposed using a medial approach and soft tissue wasreflected. The joint surface and the anterior and most posteriorextent of the tibial plateau were identified. A K-wire was inserted10 mm distal to the joint line and 12 mm anterior to the mostposterior aspect of the tibia. An 11 mm cylindrical defect wascreated using the aforementioned cannulated reamer, drilling frommedial to lateral to a depth of 25 mm without lateral corticalpenetration. The defect was rinsed copiously with saline andpacked with gauze to control bleeding. The defect was filled withthe appropriate graft material either with or without PRP and thegraft gently compressed with a bone tamp. Soft tissues were closedin layers.

Follow up and extraction of specimens

Postoperatively, the animals were housed in individual pens. Inall cases the animals were permitted to walk immediately aftersurgery. Body temperature, body weight and general conditionswere examined until completed wound healing. After 6 weeks theanimals were killed according to ethical standards by an overdoseof sodium pentobarbital 3% (Eutha 77, Essex Pharma GmbH,Munich, Germany). The proximal tibia was dissected (from thedistal femoral shaft to the proximal tibial shaft) and the soft tissuesremoved. All specimens were fixed in 10% neutral bufferedformalin solution for 14 days.

Radiological evaluation

Conventional X-rays in two planes were obtained on theexplanted tibial bones after 6 weeks (Heliodent DS, Sirona DentalSystems GmbH, Bensheim, Germany). All X-rays were evaluated bya sole experienced radiologist masked to the specific experimentalconditions. According to Sarkar et al.25 the degree of new boneformation was estimated using a semi-quantitative score from 0(no mineralised bone) up to at best 4 (complete bridging of thedefect with mineralised bone). The other score values were definedas follows: few and isolated centres of ossification (1); more, butstill discontinuous new bone formation (2); beginning, butincomplete bridging of the defect (3).

Preparation of platelet-rich plasma (PRP)

PRP was prepared from 120 ml citrated autologous whole bloodof the 8 mini-pigs allocated to the PRP group. The blood wasretrieved from the jugular vein of the mini-pig directly prior tosurgery. PRP was prepared with the ‘‘GPS1 II Platelet SeparationSystem’’ (Biomet Biologics, Warsaw, IN USA). Two 60 ml syringeswere filled with each 5 ml of citrate anticoagulant and 55 ml ofautologous whole blood. This mixture was centrifuged with aspeed of 3200 rpm for 15 min and the platelet-poor plasma (PPP)was separated from the PRP. To gain the necessary autologousthrombin for the initiation of the PRP two 10 ml syringes werefilled with 7 ml of the whole blood and centrifuged with the samespeed for 150 s. This autologous thrombin was used in combina-

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723 719

tion of 2 ml of 10% calcium chloride. Hence 20 ml of PRP weregained. Samples of PRP and native blood were analysed in anautomatic counter (ADVIAs 120, Bayer Diagnostics GmbH,Leverkusen, Germany) using veterinary software adapted to thesize of pig blood cells. The concentration of PDGF and TGFb1 werealso quantified in serum, plasma and in PRP using the ELISA-Technique (Quantikine1 ELISA-Kits, R&D Systems, MN, USA) foreach animal.

Histological preparation

The specimens were dehydrated using ascending grades ofalcohol and xylene, infiltrated, and embedded in methylmetha-crylate (Technovit 9100 NEU, Heraeus Kulzer, Wehrheim, Ger-many) for non-decalcified sectioning. During this procedure, anynegative influence of polymerisation heat was avoided byperforming controlled polymerisation in a cold atmosphere(�4 8C). After 20 h the specimens were completely polymerised.Each specimen was cut in the axial direction using a diamond wiresaw (Exakt1, Apparatebau, Norderstedt, Germany). Serial sectionswere prepared from the central parts of the defect areas, resultingin three sections each of approximately 320 mm thickness.8,28 Allspecimens were glued with acrylic cement (Technovit 7210 VLC,Heraeus Kulzer) to oversize plastic slides (size:50 mm � 100 mm � 2 mm, Dia-Plus, Walter Messner GmbH,Oststeinbeck, Germany) and ground to a final thickness ofapproximately 60 mm. All the sections were scheduled forhistomorphometric analysis and stained with toluidine blue.

Histomorphometrical analysis

Histomorphometrical analyses as well as microscopic observa-tions were performed by one experienced investigator masked tothe specific experimental conditions. For image acquisition acolour CCD camera (Colour View III, Olympus, Hamburg, Germany)was mounted on a binocular light microscope (Olympus SZ 61,Olympus). Digital images (original magnification � 6.7) wereevaluated using a software program (Cell D, Olympus GmbH,Hamburg, Germany). The quantitative analysis of new boneformation (area of new bone formation in mm2 and percentageof total new bone formation) was measured histomorphometri-cally in two predefined regions of interest. One was positioned inthe cortical defect zone and the other one in the central area of thedefect zone (Fig. 1). Three sections were evaluated for each bonedefect. Additionally, the amount of osseous bridging in the corticaldefect zone as well as the osseous bridging in the central area of thedefect zone were evaluated under standard light microscopy

Fig. 1. Scheme of a histological slide with the bone defect in the proximal tibia. The

quantitative analysis of new bone formation was measured histomorphometrically

in two regions of interest. The cortical defect zone (size: 1500 � 7000 pixels). The

central defect zone (size: 3000 � 7000 pixels).

(original magnification � 20) using a semi-quantitative score from0 (no bridging), 1 (incomplete bridging) up to at best 2 (completebridging of the defect with mineralised bone).

Statistical analysis

The statistical analysis was performed using a commerciallyavailable software program (SPSS 17.0, SPSS Inc., Chicago, IL, USA).Mean values and standard deviations were calculated for eachgroup. For the statistical comparisons between groups, the unpairedt-test was used. Significance was defined as a p-value< 0.05. In

Fig. 2. Semi-quantitative radiological evaluation. (a) X-rays of the defect area

showing a complete osseous bridging (score = 4), applying autologous cancellous

graft in combination with PRP. (b) X-rays of the defect area showing a beginning,

but still incomplete osseous bridging (score = 3), applying autologous cancellous

graft.

Fig. 3. Quantification of platelets, PDGF-bb and TGF-b1 in native blood, serum, plasma and PRP. (a) Quantification of platelets in native blood and PRP, mean values and

standard deviation are shown, p < 0.001 PRP versus native blood. (b) Quantification of PDGF-bb in serum and PRP, mean values and standard deviation are shown, p < 0.001

PRP versus serum. (c) Quantification of TGF-b1 in plasma and PRP, mean values and standard deviation are shown, p < 0.001 PRP versus plasma.

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723720

addition statistical power was evaluated (G*Power Version 3.0.10,Franz Faul, University of Kiel, Germany).

Results

The postoperative healing was uneventful in all pigs. Nocomplications such as allergic reactions, abscesses or infectionswere observed throughout the study period.

Fig. 4. Histomorphometrical analysis of the ratio of new bone concerning the central area

new bone was significantly higher in the PRP group (central area of the defect zone: p < 0

control group).

Radiological evaluation

Evaluating the X-rays areas of mineralisation were visible after6 weeks in all defects of the control as well as the PRP group.Complete osseous bridging (score = 4) was observed in three mini-pigs of the control group and in five of the PRP group. All otheranimals of both groups showed a beginning, but still incompletebridging of the defect (score = 3) (Fig. 2). Altogether there were no

of the defect zone as well as the cortical defect zone. In both defect zones the area of

.02 PRP group versus control group, cortical defect zone: (p < 0.01) PRP group versus

Fig. 5. Histological sections of the control and PRP group 6 weeks after surgery (original magnification � 6.7). Only defects of the PRP group (a) regenerated entirely. (a) PRP

group. (b) Control group.

Fig. 6. Semi-quantitative analysis of osseous bridging. (a) Semi-quantitative

analysis of the cortical defect zone. (b) Semi-quantitative analysis of the central

defect zone.

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723 721

relevant differences between the PRP and the control group in theoverall bone formation in the defect after a period of 6 weeks.

Platelet concentration and growth factor concentration in PRP

Platelet concentrations in the native blood, which was collectedjust before the operation, varied inter-individually between 364and 522 � 103/mm3 platelets (mean 433.6 � 103/mm3 � 56.4). Inthe PRP samples the platelet concentration ranged between 1735 and3330 � 103/mm3 platelets (mean 2120.3 � 103/mm3 � 550.7). Thiswas significantly higher (p < 0.001, statistical power: 0.99) than inthe native blood. On average a 4.9-fold concentration increase couldbe achieved. Comparing native blood and PRP an enrichment ofgrowth factors was detectable in the PRP. PDGF-bb in serum of nativeblood was between 15 and 514 pg/ml (mean 173.8 pg/ml � 206.3)and in PRP between 13561.3 and 22577.5 pg/ml (mean 17091.6 pg/ml � 3031.9). TGF-b1 in plasma of native blood showed a rangebetween 1187.5 and 7616.5 pg/ml (mean 3652.5 pg/ml � 2317.2)and in PRP a range between 23625.7 and 46745.4 pg/ml (mean39005.7 pg/ml � 6954.9). Hence, the concentration of PDGF-bb wassignificantly (p < 0.001, statistical power: 0.99) increased by 116.5-fold as well as the concentration of TGF-b1 significantly (p < 0.001,statistical power: 0.99) by 7.9-fold (Fig. 3a–c).

Histological results

Histomorphometrical analysis after 6 weeks of healing revealedthat the area of new bone was significantly superior in the PRPgroup concerning the central area of the defect zone (p < 0.02,statistical power: 0.87) as well as the cortical defect zone (p < 0.01,statistical power: 0.95) (Fig. 4).

All defects showed substantial new bone formation, but onlydefects of the PRP group regenerated entirely (Fig. 5). In general,the histological findings were in accordance with the results of thehistomorphometrical analysis and the X-rays. There was regularbone healing in all animals of both groups. The sections stainedwith toluidine blue showed a physiological bone remodelling in allspecimens. The presence of multinucleated giant cells/macro-phages was more prominent in the PRP group. At the margins ofthe central area of the defect zone both resorption and new boneformation could be observed more intensively in the PRP group andstronger remodelling was seen in the adjoining cortical bone. ThePRP group was superior to the control group even in the semi-quantitative assessment of the osseous bridging in both observedareas of the defect (Fig. 6). No inflammatory reaction was found inrelation to any of the grafting materials.

Discussion

Since the bone regeneration with pigs (1.2–1.5 mm/day) is verysimilar to that of humans (1.0–1.5 mm/day) we consider mini-pigsas laboratory animals on account of the best possible biologicalanalogy and transfer of experimental findings.27 The semi-quantitative analysis of the radiological findings of metaphysealdefects of the proximal tibiae of mini-pigs showed in theautologous cancellous graft group in most of the cases a beginning,but incomplete bridging of the defects after 6 weeks, whereas thecomplete osseous bridging of the defects could be observed morefrequently within the PRP group. The histomorphometricalanalysis of the defect zones did show for both groups a proper

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723722

bone regeneration. This was in the PRP group in the central as wellas in the cortical defect zones significantly higher than in thecontrol group.

The multitude of clinical and experimental studies publishedabout the application of PRP as a carrier of osteoinductive growthfactors report on a considerable acceleration of the bone healingafter using PRP.5,6,16,20,31,34 The aim of using PRP to improve bonehealing is caused by releasing of growth factors, which areincluded in the platelets. In contrast to other osteoinductive agentslike BMP-2 and BMP-7, PRP with its autologous origin does notcause any risk of allergies and graft versus host reactions.19 Thegrowth factors contained in PRP are PDGF, TGFb, VEGF, EGF, IGF-1,and PF-4.20,31,34 The in vitro experiments demonstrated that eachof these factors has different effects on the bone healing.32 Themost important specific activities of PDGF include mitogenesis,angiogenesis and macrophage activation, whereas these are notonly responsible for the healing process, they also serve as asecondary source for PDGF to continue bone repair and boneregeneration. The importance of PDGF also becomes evident due toits numerous appearance in platelets.4,29 TGFb is produced inplatelets and macrophages.22 It acts as a paracrine growth factor(activating adjacent cells, especially fibroblasts, marrow stem cellsand the preosteoblasts, which can produce their own TGFb) and asan autocrine growth factor. This is why TGFb represents amechanism for sustaining a long-term healing and bone regener-ation module and even evolve into a bone remodelling factor overtime.3 The most important functions of TGFb seem to bechemotaxis and mitogenesis of osteoblasts.24 In addition TGFbinhibits the formation of osteoclasts and bone resorption.23

Present research shows that the enrichment of PRP can influencethe early stage of bone healing, whereas this influence graduallydiminishes.30 Platelets have a life span of about 5 days. Theinfluence of platelets in the context of bone healing works with twomechanisms, first through an increased number of stem cells andtheir activation, which release TGFb on their own, and secondthrough chemotaxis and the activation of macrophages, which takeover the function of the platelets within the process of bonehealing.24 After 4–6 weeks the oxygen gradient, which is neededfor the activities of the macrophages, is nullified due torevascularisation, so that the macrophages leaves the defect area.The transplant then becomes self-sustaining.17

These mechanisms serve as an explanation for the results of thisstudy. Thereby we determined that the combined use of PRP andautologous cancellous bone, which contains the correspondingreceptors for PDGF and TGFb,20 optimises the healing of bonedefects of the proximal tibia of mini-pigs compared to the isolateduse of autologous cancellous bone.

These results are in contrast to the results of several otherstudies.1,7,10,11,15,25 These studies, however, lack a positive effect ofPRP, whereby these authors did not use a combination of PRP andautologous cancellous bone. This could explain differingresults.7,10,11,15,25 This decisive intensification of the effects ofPRP through applying a combination of it with autologouscancellous bone is also described by Thor et al.30, who also pointout the increased effects of growth factors through the existence ofthe corresponding receptors in autologous cancellous bone.20

Indeed Aghaloo et al.1 state a lacking improvement in the healingof bone defects using autologous cancellous graft combined withPRP compared to its isolated use. In this very study, however, the PRPpreparation method resulted in a platelet concentration that was 7.3times higher than in native blood. The platelet concentration neededfor a positive PRP effect on the bone regeneration appears to bewithin a narrow span of concentration. A platelet concentration thatis 3–5 times higher to the concentration in plasma seems to beespecially favourable for bone regeneration. Below this concentra-tion the effect of PRP is suboptimal and, paradoxically, a higher

concentration leads to an inhibiting effect on bone regeneration.32

Our method used for the preparation of PRP enabled us to reach aplatelet concentration that averaged 4.9 times the regular amount,whereby we were able to reach an optimal concentration for apossibly positive PRP effect. Furthermore, within the context of ourquantitative regulations we were able to note a significantly higherconcentration of the important growth factors PDGF and TGFb in theso prepared PRP compared to native blood.

Another reason for the results of our study seems to lie in thechoice of the localisation for the critical size defects. For, especiallythrough the use of autologous cancellous bone, the tendency for acomplete recovery of a long bone defect is distinctly higher in themetaphyseal area than in the diaphyseal area. Accordingly, Sarkaret al.25 stated that the use of PRP in combination with a collagenscaffold for the treatment of a critical size defect in the diaphysealarea of a load-bearing bone of a sheep showed no increase in thebone regeneration. Therefore, in order to clarify the effectiveness ofPRP in the treatment of critical size defects in the diaphyseal area ofa load-bearing long bone further studies should be carried out withthe aim of a combined use of PRP and autologous cancellous bone.

Conclusion

This study demonstrates that PRP combined with autologouscancellous bone leads to a significantly better bone regenerationcompared to isolated application of autologous cancellous bone inan in vivo critical size defect on load-bearing long bones of mini-pigs during our chosen time frame of 6 weeks. In contrast to otherstudies our PRP preparation method resulted in an increasedconcentration of both platelets and growth factors. Furthermorethe preparation and application of PRP represent an easy andsuccessful method to promote bone healing in this model. Due toits autologous origin PRP is an attractive alternative to otherosteoinductive agents like BMP-2 and BMP-7 without the risk ofallergies and graft versus host reactions.19 Besides, the use of PRPdoes not result in essential additional costs.

Conflict of interest statement

The authors confirm that there is no financial conflict of interestin this study.

References

1. Aghaloo TL, Moy PK, Freymiller EG. Investigation of platelet-rich plasma inrabbit cranial defects: a pilot study. J Oral Maxillofac Surg 2002;60:1176–81.

2. Antoniades HN. Human platelet-derived growth factor (PDGF): purification ofPDGF-I and PDGF-II and separation of their reduced subunits. Proc Natl Acad SciUSA 1981;78:7314–7.

3. Beck LS, DeGuzman L, Lee WP, et al. One systemic administration of transform-ing growth factor-beta 1 reverses age- or glucocorticoid-impaired woundhealing. J Clin Invest 1993;92:2841–9.

4. Bowen-Pope DF, Vogel A, Ross R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor recep-tors accompany transformation by a wide spectrum of agents. Proc Natl AcadSci USA 1984;81:2396–400.

5. Camargo PM, Lekovic V, Weinlaender M, et al. Platelet-rich plasma and bovineporous bone mineral combined with guided tissue regeneration in the treat-ment of intrabony defects in humans. J Periodontal Res 2002;37:300–6.

6. Dallari D, Savarino L, Stagni C, et al. Enhanced tibial osteotomy healing with useof bone grafts supplemented with platelet gel or platelet gel and bone marrowstromal cells. J Bone Joint Surg Am 2007;89:2413–20.

7. de Vasconcelos Gurgel BC, Goncalves PF, Pimentel SP, et al. Platelet-rich plasmamay not provide any additional effect when associated with guided boneregeneration around dental implants in dogs. Clin Oral Implants Res2007;18:649–54.

8. Donath K. The diagnostic value of the new method for the study of undecalcifiedbones and teeth with attached soft tissue (Sage-Schliff (sawing and grinding)technique). Pathol Res Pract 1985;179:631–3.

9. Friedlaender GE. Osteogenic protein-1 in treatment of tibial nonunions: currentstatus. Surg Technol Int 2004;13:249–52.

10. Fuerst G, Gruber R, Tangl S, et al. Effects of fibrin sealant protein concentratewith and without platelet-released growth factors on bony healing of cortical

M. Hakimi et al. / Injury, Int. J. Care Injured 41 (2010) 717–723 723

mandibular defects. An experimental study in minipigs. Clin Oral Implants Res2004;15:301–7.

11. Fuerst G, Reinhard G, Tangl S, et al. Effect of platelet-released growth factors andcollagen type I on osseous regeneration of mandibular defects. A pilot study inminipigs. J Clin Periodontol 2004;31:784–90.

12. Garg AK. The use of platelet-rich plasma to enhance the success of bone graftsaround dental implants. Dent Implantol Update 2000;11:17–21.

13. Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury2005;36(Suppl. 3):S20–27.

14. Govender S, Csimma C, Genant HK, et al. Recombinant human bone morpho-genetic protein-2 for treatment of open tibial fractures: a prospective, con-trolled, randomized study of four hundred and fifty patients. J Bone Joint SurgAm 2002;84-A:2123–34.

15. Jensen TB, Rahbek O, Overgaard S, Soballe K. Platelet rich plasma and freshfrozen bone allograft as enhancement of implant fixation. An experimentalstudy in dogs. J Orthop Res 2004;22:653–8.

16. Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of marrow-derivedmesenchymal stem cells and platelet-rich plasma during distraction osteogen-esis—a preliminary result of three cases. Bone 2004;35:892–8.

17. Knighton DR, Silver IA, Hunt TK. Regulation of wound-healing angiogenesis-effect of oxygen gradients and inspired oxygen concentration. Surgery1981;90:262–70.

18. Mahendra A, Maclean AD. Available biological treatments for complex non-unions. Injury 2007;38(Suppl. 4):S7–12.

19. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral MaxillofacSurg 2004;62:489–96.

20. Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: growth factorenhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod1998;85:638–46.

21. Marx RE, Ehler WJ, Peleg M. ‘‘Mandibular and facial reconstruction’’ rehabilita-tion of the head and neck cancer patient. Bone 1996;19:59S–82S.

22. Miyazono K, Heldin CH. The mechanism of action of transforming growthfactor-beta. Gastroenterol Jpn 1993;28(Suppl. 4):81–5 [discussion 86–7].

23. Mohan S, Baylink DJ. Bone growth factors. Clin Orthop Relat Res 1991;30–48.24. Pierce GF, Tarpley JE, Yanagihara D, et al. Platelet-derived growth factor (BB

homodimer), transforming growth factor-beta 1, and basic fibroblast growth

factor in dermal wound healing. Neovessel and matrix formation and cessationof repair. Am J Pathol 1992;140:1375–88.

25. Sarkar MR, Augat P, Shefelbine SJ, et al. Bone formation in a long bone defectmodel using a platelet-rich plasma-loaded collagen scaffold. Biomaterials2006;27:1817–23.

26. Schieker M, Heiss C, Mutschler W. Bone substitutes. Unfallchirurg 2008;111:613–9 [quiz 620].

27. Schlegel KA, Kloss FR, Schultze-Mosgau S, et al. Osseous defect regenerationusing autogenous bone alone or combined with Biogran or Algipore with andwithout added thrombocytes. A microradiologic evaluation. Mund KieferGesichtschir 2003;7:112–8.

28. Schwarz F, Herten M, Ferrari D, et al. Guided bone regeneration at dehiscence-type defects using biphasic hydroxyapatite + beta tricalcium phosphate (BoneCeramic) or a collagen-coated natural bone mineral (BioOss Collagen): animmunohistochemical study in dogs. Int J Oral Maxillofac Surg 2007;36:1198–206.

29. Singh JP, Chaikin MA, Stiles CD. Phylogenetic analysis of platelet-derivedgrowth factor by radio-receptor assay. J Cell Biol 1982;95:667–71.

30. Thor A, Franke-Stenport V, Johansson CB, Rasmusson L. Early bone formation inhuman bone grafts treated with platelet-rich plasma: preliminary histomor-phometric results. Int J Oral Maxillofac Surg 2007;36:1164–71.

31. Veillette CJ, McKee MD. Growth factors—BMPs, DBMs, and buffy coat products:are there any proven differences amongst them? Injury 2007;38(Suppl. 1):S38–48.

32. Weibrich G, Gnoth SH, Otto M, et al. Growth stimulation of human osteoblast-like cells by thrombocyte concentrates in vitro. Mund Kiefer Gesichtschir2002;6:168–74.

33. Wheeler DL, Cross AR, Eschbach EJ, et al. Grafting of massive tibial subchondralbone defects in a caprine model using beta-tricalcium phosphate versus auto-graft. J Orthop Trauma 2005;19:85–91.

34. Wrotniak M, Bielecki T, Gazdzik TS. Current opinion about using the platelet-rich gel in orthopaedics and trauma surgery. Ortop Traumatol Rehabil2007;9:227–38.

35. Zimmermann G, Moghaddam A, Wagner C, et al. Clinical experience with bonemorphogenetic protein 7 (BMP 7) in nonunions of long bones. Unfallchirurg2006;109:528–37.