J Periodontol • August 2005

1287

* Laboratory for Applied Periodontal and Craniofacial Regeneration, Department ofPeriodontology, Temple University School of Dentistry, Philadelphia, PA.

† Division of Oral and Maxillofacial Radiology, Department of Oral and MaxillofacialPathology, Medicine and Surgery, Temple University School of Dentistry.

‡ Department of Periodontology and Dental Hygiene, University of Detroit Mercy School ofDentistry, Detroit, MI.

§ Central Animal Facility, Temple University School of Medicine.� Department of Physiology, Temple University School of Medicine.

Platelets have been identified as arich source of growth factors thatinclude platelet-derived growth fac-

tor (PDGF) and transforming growth fac-tor-beta (TGF-β). PDGF and TGF-β arestored in platelet α-granules and emergefrom degranulating platelets at initiationof wound healing.1,2 PDGF plays animportant role in stimulating mitogene-sis, angiogenesis, and macrophage acti-vation while acting as a chemoattractantfor osteoblast and fibroblast precursorcells in mesenchymal tissues to initiateconnective tissue healing.2,3 TGF-βplays a central role in bone repair mech-anisms as it stimulates chemotaxis andmitogenesis of osteoblast precursorcells.4 Moreover, TGF-β stimulatesosteoblasts and fibroblasts to depositcollagen into the extracellular matrix forconnective tissue healing and bone for-mation, as well as preventing osteoclastdifferentiation and bone resorption.1,5

Platelet-rich plasma (PRP) is obtainedby sequestering blood into three com-ponents by gradient density centrifuga-tion to include red blood cells (bottomlayer), platelet-rich plasma (middlelayer), and platelet-poor plasma (toplayer).6,7 It is prepared with the intent toinfluence soft and hard tissue repairand/or regeneration through the stimu-lation and regulation of cellular events.8

Reported clinical benefits of PRP includeimproved hemostasis, ease of manipu-lation due to its gelatinous adhesive con-sistency, and minimal concern for riskof infection, disease transmission, and

Analysis of Rat Calvaria DefectsImplanted With a Platelet-Rich PlasmaPreparation: Radiographic ObservationsMary E. Pryor,* Jie Yang,† Giuseppe Polimeni,* Ki-Tae Koo,* Michael J. Hartman,* Howard Gross,‡Alexis Agelan,§ Joanne M. Manns,� and Ulf M.E. Wikesjö*

Background: Platelet-rich plasma (PRP) harbors growth factorsidentified in bone. It has been suggested that these factors enhanceosteogenesis. The objective of this study was to conduct a radio-graphic evaluation on local bone formation following surgicalimplantation of a PRP preparation using a critical-size rat calvariadefect model.

Methods: Thirty 22-week-old male Sprague-Dawley rats were used.The PRP preparation was obtained from 10 ml of whole blood drawnfrom one age-matched donor rat. The preparation was processed bygradient density centrifugation and stored at −80°C until use. Usingaseptic techniques, the PRP preparation soak-loaded onto anabsorbable collagen sponge (ACS) carrier or ACS alone was surgi-cally implanted into contralateral critical-size 6 mm rat calvariaosteotomies in 18 animals. Twelve animals received ACS alone ver-sus sham surgery in contralateral defects. Animals were sacrificed at4 and 8 weeks when biopsies were collected and radiographs wereobtained using a standardized protocol. Three masked examinersindependently evaluated the radiographic images of the defect sites.Examiner reproducibility was examined by repeat evaluation of alldefect sites (r = 0.6; P <0.0001).

Results: The animals were maintained without adverse events.Defect sites in two animals receiving ACS versus sham surgery(4-week healing interval) were not evaluated due to specimen damage.Seventy-five percent of the sites (PRP/ACS or ACS) exhibited partialclosure at 4 weeks; one site (ACS) exhibited full closure without sig-nificant differences between protocols (P = 0.1797). Fifty percent ofthe sites receiving PRP/ACS exhibited full closure and 20% partial clo-sure at 8 weeks versus 20% and 80%, respectively, for the ACS con-trol (P = 0.7532). There were no noteworthy differences between sitesreceiving ACS versus sham surgery at 4 or 8 weeks.

Conclusion: The results suggest that the PRP preparation doesnot have a significant effect on osteogenesis. J Periodontol 2005;76:1287-1292.

KEY WORDSAnimal studies; bone and bones; growth factors; osteogenesis;plasma, platelet-rich; tissue engineering.

40339.qxd 8/8/05 7:32 AM Page 1287

1288

Effect of PRP on Rat Calvaria Defects Volume 76 • Number 8

immunogenic reactions as it is anautologous preparation.6,8,9

Animal models have beenemployed to evaluate the regener-ative potential of PRP for peri-implant bone healing,10-13 sinusaugmentation combined with bovinehydroxyapatite14 or autogenousbone,15 for mandibular resec-tion,16,17 and cranial defects com-bined with autogenous bone18 oranorganic bovine bone.19,20 Theresults from these studies indicatethat the use of PRP in conjunctionwith bone derivative/substitute(s)may or may not enhance local bone formation and mat-uration.

Radiographic observations of osseous defects fol-lowing implantation of PRP and/or bone biomaterialssuggest a 2-fold increase in bone density at 2, 4, and6 months when PRP was added to autologous cancel-lous cellular bone marrow implanted into large (5 cm)mandibular resection defects.7 Similar results havebeen shown in 15 mm rabbit calvaria defects implantedwith a PRP/natural cancellous bovine bone mineralconstruct.19 More mineralized areas were observed insites receiving the PRP/bovine bone combination thanin controls at 4 and 8 weeks post-implantation. In addi-tion, Fennis et al.16 reported accelerated bone healingin mandibular osteotomy sites with no signs of radio-lucent areas at 6 weeks post-surgery following implan-tation a PRP/autogenous bone construct. These studiessuggest a beneficial effect by adding PRP to autoge-nous bone or bone biomaterials to promote local boneformation; however, a genuine effect of the PRP prepa-rations on osteogenesis has not been demonstrated.The objective of this study was to conduct a radio-graphic evaluation on bone formation following sur-gical implantation of a PRP preparation using acritical-size rat calvaria defect model.

MATERIALS AND METHODSAnimalsThirty 22-week-old male Sprague-Dawley rats, weightapproximately 525 g, were used following a protocolapproved by the Institutional Animal Care and Use Com-mittee, Temple University, Philadelphia, Pennsylvania.The animals were individually housed in plastic cagesin a monitored environment (21°C; 12:12 light cycle).They had ad libitum access to drinking water and astandard laboratory rat food pellet diet. The animalswere monitored for signs of infection and discomfortpre- and post-surgery until euthanasia.

Platelet-Rich Plasma PreparationThe PRP preparation was obtained from one age-matched Sprague-Dawley rat. The animal was anes-

thetized using isoflurane inhalation anesthesia¶ (4% to5% induction; 2% to 3% maintenance). An aorta heartpuncture with complete exsanguination was performedusing 5 ml vacutainer tubes containing 0.84 ml acid-citrate-dextrose (ACD) and 10 mM EDTA. A total of10 ml of whole blood was obtained. The blood wastransferred to a centrifuge tube and spun at 450 g for30 minutes. The PRP was removed and placed into anew tube. An equal volume of Tyrode’s buffer (137 mMNaCl, 2.7 mM KCl, 2 mM MgCl, 0.5 mM NaH2PO4,5 mM glucose, 10 mM HEPES, 0.2% BSA; pH 7.4)was added to the PRP. The diluted PRP was centrifugedat 1,400 g for 15 minutes to pellet the platelets. Theplatelet pellet was resuspended in 2 ml Tyrode’s buffer.Platelet counts were determined from this preparationusing a Coulter counter. Ten µl of 20% Triton X-100(final concentration 0.1%) was added to lyse the cells.As soon as the solution cleared, the lysate (PRP prepa-ration) was placed into 1.5 ml microcentrifuge tubesand spun at 16,000 g for 15 minutes to pellet cell debris.All procedures were performed at room temperature.The cell lysate was aliquoted and stored at −80°C.

Experimental SurgeryAnesthesia and pain control followed recommendedroutines for the species. The animals were anesthetizedusing isoflurane inhalation anesthesia (4% to 5% induc-tion; 2% to 3% maintenance). Buprenorphine HCl, 0.02to 0.03 mg/kg was administered presurgically. Priorto surgery, the animal’s head was shaved, washed witha disinfectant, and stabilized by a nose cone appara-tus. A midline incision was made from the nasofrontalarea to the external occipital protuberance along themid-sagittal suture. The skin and underlying tissues,including the temporal muscle, were reflected bilater-ally to expose the full extent of the calvaria (Fig. 1).

In each animal, one calvarial through-and-throughosteotomy, 6.0 mm in diameter, was trephined into thedorsal portion of the parietal bone on each side of themid-sagittal suture using a dental handpiece and a

Figure 1.Bilateral craniotomy defects before (left) and after (right) implantation of PRP/ACS and ACS.(Reprinted with permission from Blackwell Publishing.21)

¶ E-Z Anesthesia, Euthanex Corp., Palmer, PA.

40339.qxd 8/8/05 7:32 AM Page 1288

1289

J Periodontol • August 2005 Pryor, Yang, Polimeni, et al.

trephine bur# under constant irrigation of sterile saline.The trephined bone was removed from the surgical field.Using aseptic techniques, 42 µl of the PRP preparationwas used to soak-load a precut (diameter 6 × 1.5 mm)absorbable collagen sponge (ACS),** which was usedas a carrier. The PRP/ACS construct or ACS alone wasimplanted into contralateral calvarial osteotomies in 18animals. Twelve animals received ACS without the PRPpreparation versus sham surgery in contralateral cal-varial osteotomies. The skin and underlying tissues werereadapted to cover the exposed calvarium. The marginsof the wound were closed using autoclips.†† A bacitracin-neomycin-polymyxin antibiotic ointment‡‡ was topicallyapplied to the eyes and the animal was monitored untilanesthesia recovery. Animals were sacrificed at 4 and8 weeks post-surgery by CO2 inhalation. The cranialbone including the bilateral defects was removed in total,rinsed in water, and placed into 10% buffered formalin.

In all, four experimental groups were constituted.According to the experimental design, the animals wererandomized to receive one of the following protocols:1) PRP/ACS versus ACS (10 animals; observation inter-val 8 weeks); 2) PRP/ACS versus ACS (eight animals;observation interval 4 weeks); 3) ACS versus shamsurgery (seven animals; observation 8 weeks); or 4) ACSversus sham surgery (five animals; observation 4 weeks).

Radiographic Processing and EvaluationStandardized radiographic images of the rat calvariagross specimens were obtained using a dental radio-graphic unit (70 kVp, 7 mA for 0.083 seconds)§§ andx-ray film.� � The distance between the x-ray source andfilms was 12 inches. Radiographs were processed in anautomatic dental film processor.¶¶ The radiographs(analog films) were transformed into digitized imagesusing a film scanner## at 1,200 dpi, which was con-nected to a standard personal computer. There was noattempt to adjust or calibrate the optical density of theradiographic images. The distinction between mineral-ized and non-mineralized tissues was made for eachdefect by visual evaluation of the gray scale in eachimage by three masked examiners (investigators MEP,JY, and UMEW). The defects were classified accordingto the following criteria: 1) no closure: the bone defectremained radiolucent with the exception of limited newbone apposition at the defect margins; 2) partial clo-sure: the bone defect was partially radiopaque, exhibit-ing regions of radiolucencies and radiopacitiessuggestive of new bone formation from the defect mar-gins without establishing bone continuity; or 3) completeclosure: the bone defect showed radiopacity/osseouscontinuity between the defect margins.

Complete agreement was reached most of the time.When the classification of the defect was not unani-mous, the defect was reevaluated to achieve consensusby two or all three evaluators.

Statistical AnalysisThe Wilcoxon sign rank test was used to evaluate dif-ferences between protocols within experimental groups.The level of significance was set at 5%. Examinerreproducibility was examined by repeat evaluation ofall defect sites (r = 0.6; P <0.0001).

RESULTSAll of the animals were maintained without significantadverse events. A platelet count of 1.76 × 109 cells/mlwas obtained from the PRP preparation. Two ACS ver-sus sham surgery specimens (4-week healing inter-val) were not evaluated due to damage.

PRP/ACS versus ACSResults of the radiographic evaluation and represen-tative radiographs of sites receiving PRP/ACS versusACS are shown in Table 1 and Figure 2. At 4 weeks,75% of the sites, PRP/ACS or ACS, exhibited partialclosure. Complete closure was observed in one of eight(12.5%) sites receiving ACS. No closure was observedin two of eight (25%) and one of eight (12.5%) sitesreceiving PRP/ACS or ACS, respectively. At 8 weeks,two of 10 (20%) and eight of 10 (80%) of the sitesreceiving PRP/ACS or ACS, respectively, exhibited par-tial closure. Complete closure was observed in five of10 (50%) sites implanted with PRP/ACS and in two of10 (20%) sites receiving ACS. No closure was observedin three of 10 (30%) sites receiving PRP/ACS. Therewere no statistically significant differences in defectclosure between treatments in animals receivingPRP/ACS versus ACS at 4 (P = 0.1797) or 8 (P =0.7532) weeks.

# 11-31-0050, Ace Surgical Supply, Inc., Brockton, MA.** CollaCote, Sulzer Dental Inc., Carlsbad, CA.†† Autoclip Wound Closing System, Stoelting Co., Wood Dale, IL.‡‡ Vetropolycin Ophthalmic Ointment, Pharmaderm, Melville, NY.§§ Gendex 770, Gendex Corporation, Des Plaines, IL.� � #2 Ultra Speed, Eastman Kodak Company, Rochester, NY.¶¶ A/T 2000, Air Techniques, Hicksville, NY.## 620ST, AcerScan, San Jose, CA.

Table 1.

Radiographic Evaluation of Bone Fill inContralateral Craniotomy Defects ReceivingPRP/ACS or ACS

PRP/ACS ACS

4 Weeks 8 Weeks 4 Weeks 8 Weeks Closure (N = 8) (N = 10) (N = 8) (N = 10)

No closure 2 3 1 0

Partial closure 6 2 6 8

Complete closure 0 5 1 2

40339.qxd 8/8/05 7:32 AM Page 1289

1290

Effect of PRP on Rat Calvaria Defects Volume 76 • Number 8

Table 2.

Radiographic Evaluation of Bone Fill inContralateral Craniotomy Defects ReceivingACS or Sham Surgery

ACS Sham Surgery

4 Weeks 8 Weeks 4 Weeks 8 Weeks Closure (N = 3) (N = 7) (N = 3) (N = 7)

No closure 0 1 0 0

Partial closure 1 1 1 1

Complete closure 2 5 2 6

ACS versus Sham SurgeryResults of the radiographic evaluation and represen-tative radiographic images of the defect sites receiv-ing ACS versus sham surgery are shown in Table 2 andFigure 2. At 4 weeks, 33% of the sites receiving ACSor sham surgery exhibited partial closure. Completeclosure was observed in two of three (67%) sites receiv-ing ACS and in two of three (67%) sites receiving shamsurgery. At 8 weeks, 14% of the sites receiving ACSor sham surgery exhibited partial closure. Completeclosure was observed in five of seven (72%) sitesreceiving ACS and in six of seven (86%) sites receiv-ing sham surgery. No closure was observed in one ofseven (14%) of the sites receiving ACS. In all, therewere no noteworthy differences between sites receiv-ing ACS versus sham surgery at 4 or 8 weeks.

DISCUSSIONThe objective of this study was to evaluate localbone formation following surgical implantation of aPRP preparation. Contralateral calvarial critical-sizeosteotomy defects in 18 adult Sprague-Dawley ratswere implanted with PRP/ACS or ACS alone. Twelveanimals received ACS without the PRP preparation or

sham surgery in contralateraldefects. The defect sites were sub-jected to a radiographic analysisfollowing a 4- and 8-week healinginterval. The results suggest thatthe PRP preparation does not havea significant effect on osteogene-sis in this experimental model.

The critical-size defect possessesdimensions precluding spontaneousbone healing during the lifetime ofthe animal unless implanted withan osteogenic, osteoconductive,or osteoinductive technology.22,23

Schmitz and Hollinger22 suggestedthat an 8 mm defect is suitable to

evaluate candidate biomaterials for bone regenerationand constitutes a critical-size defect in the rat. Thisexperimental design has been utilized in previous stud-ies to evaluate the osteoconductive/inductive potentialof candidate biologics, biomaterials, and devices forbone reconstruction.24-29 Others have defined andsuccessfully used smaller critical-size rat calvarialdefects.23,30-38 Thus, the geometric and physiologicalnature of the rat calvaria and the 6 mm trephine defectsused in this study appear adequate to investigate theregenerative potential of the PRP preparation.

Radiographic evaluations have been used to evalu-ate the biologic potential of various devices, as well asosteoconductive/inductive biomaterials and biologics topromote local bone formation in rat calvarial defects. Inbrief, barrier membranes for guided bone regenera-tion,37,38 autograft bone,30 and growth and differentia-tion factors, including purified protein constructs24,26-29

or combinations thereof, have been evaluated. Theradiographic observations in these studies demonstratedlimited radiographic bone fill in the control sites. Thisis in contrast to the findings in this study where com-plete defect closure was observed in several sites receiv-ing ACS or sham surgery at 4 and 8 weeks post-surgery.

Marx et al.7 reported an increase in radiographicbone density at 2, 4, and 6 months post-surgery fol-lowing implantation of PRP/cancellous bone marrowconstructs into mandibular full-thickness resectiondefects versus cancellous bone marrow without PRP.Kim et al.19 also observed increased radiographic bonedensity at 4 and 8 weeks post-surgery followingimplantation of platelet concentrates and natural can-cellous bovine bone mineral versus natural cancellousbovine bone mineral alone in a 15 mm rabbit calvariadefect model. In contrast, the PRP preparation used inthis study did not provide evidence of enhanced localbone formation at 4 and 8 weeks.

On the other hand, Aghaloo et al.,18 utilizing 8 mmcalvaria defects to evaluate the effect of PRP on localbone formation, made similar observations to those in

Figure 2.Radiographs (8-week observations) of bilateral craniotomy defects in animals receiving PRP/ACSversus ACS and ACS versus sham surgery. A and C show defect sites that exhibit complete closure.B and D show defect sites with no closure.

40339.qxd 8/8/05 7:32 AM Page 1290

1291

J Periodontol • August 2005 Pryor, Yang, Polimeni, et al.

the present study. The calvaria defects were implantedwith autogenous bone, PRP, PRP/autogenous bone,and sham surgery. A tendency toward increased radio-graphic bone density was observed for sites receivingautogenous bone or PRP/autogenous bone comparedto PRP or sham surgery controls. However, no signif-icant differences were observed between autogenousbone and PRP/autogenous bone. There was a trendtoward less bone formation when PRP alone was com-pared to sham surgery. In a subsequent study, thedefect sites were implanted with either autogenousbone, anorganic bovine bone, or PRP/anorganic bovinebone and compared to sham surgery.20 The resultsdemonstrated no statistically significant differences inradiographic bone density among experimental sites.Collectively, these observations and those observed inthe present study suggest that PRP preparations maynot have a significant effect on local bone formation.

ACKNOWLEDGMENTSThe authors thank Lewis T. Bright, Temple University,for excellent animal technical care. This study wassupported, in part, by NIH-NCMHD grant 1-R24-MD001096-01.

REFERENCES1. Sporn MB, Roberts AB. Transforming growth factor-ß.

Multiple actions and potential clinical applications. JAMA1989;262:938-941.

2. Heldin CH, Westermark B. Mechanism of action and invivo role of platelet-derived growth factor. Physiol Rev1999;79:1283-1316.

3. Lind M. Growth factors: Possible new clinical tools. ActaOrthop Scand 1996;67:407-417.

4. Canalis E, McCarthy T, Centrella M. Growth factors andthe regulation of bone remodeling. J Clin Invest 1988;81:277-281.

5. Mohan S, Baylink DJ. Bone growth factors. Clin Orthop1991;263:30-48.

6. Whitman DH, Berry RL, Green DM. Platelet gel: An autol-ogous alternative to fibrin glue with applications in oraland maxillofacial surgery. J Oral Maxillofac Surg1997;55:1294-1299.

7. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR,Strauss JE, Georgeff KR. Platelet-rich plasma growthfactor enhancement for bone grafts. Oral Surg Oral MedOral Pathol Oral Radiol Endod 1998;85:638-646.

8. Anitua E. Plasma rich in growth factors: Preliminary resultsof use in the preparation of future sites for implants. IntJ Oral Maxillofac Implants 1999;14:529-535.

9. Gonshor A. Technique for producing platelet-rich plasmaand platelet concentrate: Background and process. IntJ Periodontics Restorative Dent 2002;22:547-557.

10. Kim S-G, Kim W-K, Park J-C, Kim H-J. A comparativestudy of osseointegration of Avana implants in a dem-ineralized freeze-dried bone alone or with platelet-richplasma. J Oral Maxillofac Surg 2002;60:1018-1025.

11. Fürst G, Gruber R, Tangl S, Sanroman F, Watzek G.Enhanced bone-to-implant contact by platelet-releasedgrowth factors in mandibular cortical bone: A histo-morphometric study in minipigs. Int J Oral MaxillofacImplants 2003;18:685-690.

12. Schlegel KA, Kloss FR, Kessler P, Schultze-Mosgau S,Nkenke E, Wiltfang J. Bone conditioning to enhanceimplant osseointegration: An experimental study in pigs.Int J Oral Maxillofac Implants 2003;18:505-511.

13. Zechner W, Tangl S, Tepper G, et al. Influence of platelet-rich plasma on osseous healing of dental implants: A his-tologic and histomorphometric study in minipigs. Int JOral Maxillofac Implants 2003;18:15-22.

14. Fürst G, Gruber R, Tangl S, et al. Sinus grafting withautogenous platelet-rich plasma and bovine hydroxya-patite. A histomorphometric study in minipigs. Clin OralImplants Res 2003;14:500-508.

15. Jakse N, Tangl S, Gilli R. et al. Influence of PRP on auto-genous sinus grafts: An experimental study on sheep.Clin Oral Implants Res 2003;14:578-583.

16. Fennis JP, Stoelinga PJ, Jansen JA. Mandibular recon-struction: A clinical and radiographic animal study onthe use of autogenous scaffolds and platelet-rich plasma.Int J Oral Maxillofac Surg 2002;31:281-286.

17. Fennis JP, Stoelinga PJ, Jansen JA. Mandibular recon-struction: A histological and histomorphometric studyon the use of autogenous scaffolds, particulate cortico-cancellous bone grafts and platelet-rich plasma in goats.Int J Oral Maxillofac Surg 2004;33:48-55.

18. Aghaloo TL, Moy PK, Freymiller EG. Investigation ofplatelet-rich plasma in rabbit cranial defects: A pilotstudy. J Oral Maxillofac Surg 2002;60:1176-1181.

19. Kim E-S, Park E-J, Choung P-H. Platelet concentrationand its effect on bone formation in calvarial defects: Anexperimental study in rabbits. J Prosthet Dent 2001;86:428-433.

20. Aghaloo TL, Moy PK, Freymiller EG. Evaluation ofplatelet-rich plasma in combination with anorganicbovine bone in the rabbit cranium: A pilot study. Int JOral Maxillofac Implants 2004;19:59-65.

21. Pryor ME, Polimeni G, Koo K-T, et al. Analysis of rat cal-varia defects implanted with a platelet-rich plasmapreparation: Histologic and histometric observations. JClin Periodontol; online publication date: June 30, 2005.DOI: 10.1111/j.1600-051X.2005.00772.x.

22. Schmitz JP, Hollinger JO. The critical size defect as anexperimental model for craniomandibulofacial nonunions.Clin Orthop Relat Res 1986;205:299-308.

23. Bosch C, Melsen B, Vargervik K. Importance of the criti-cal-size bone defect in testing bone-regenerating materi-als. J Craniofac Surg 1998;9:310-316.

24. Takagi K, Urist M. The reaction of the dura to bone mor-phogenetic protein (BMP) in repair of skull defects. AnnSurg 1982;196:100-109.

25. Dahlin C, Alberius P, Linde A. Osteopromotion for cran-ioplasty. An experimental study in rats using a mem-brane technique. J Neurosurg 1991;74:487-491.

26. Marden LJ, Fan RS, Pierce GF, Reddi AH, Hollinger JO.Platelet-derived growth factor inhibits bone regenerationinduced by osteogenin, a bone morphogenetic protein,in a rat craniotomy defects. J Clin Invest 1993;92:2897-2905.

27. Marden LJ, Hollinger JO, Chaudhari A, Turek T, SchaubRG, Ron E. Recombinant human bone morphogeneticprotein-2 is superior to demineralized bone matrix inrepairing craniotomy defects in rats. J Biomed Mater Res1994;28:1127-1138.

28. Winn SR, Schmitt JM, Buck D, Hu Y, Grainger D,Hollinger JO. Tissue-engineered bone biomimetic toregenerate calvarial critical-sized defects in athymic rats.J Biomed Mater Res 1999;45:414-421.

29. Ahn S-H, Kim C-S, Suk H-J, et al. Effect of recombinant

40339.qxd 8/8/05 7:32 AM Page 1291

1292

Effect of PRP on Rat Calvaria Defects Volume 76 • Number 8

human bone morphogenetic protein-4 with carriers in ratcalvarial defects. J Periodontol 2003;74:787-797.

30. Turnbull RS, Freeman E. Use of wounds in the parietalbone of the rat for evaluating bone marrow for graftinginto periodontal defects. J Periodontal Res 1974;9:39-43.

31. Mulliken JB, Glowacki J. Induced osteogenesis for repairand construction in the craniofacial region. Plast Recon-str Surg 1980;65:553-559.

32. Bosch C, Melsen B, Vargervik K. Guided bone regenera-tion in calvarial bone defects using polytetrafluoroethylenemembranes. Cleft Palate Craniofac J 1995;32:311-317.

33. Bosch C, Melsen B, Gibbons R, Vargervik K. Humanrecombinant transforming growth factor-β1 in healing ofcalvarial bone defects. J Craniofac Surg 1996;7:300-310.

34. Bohning BP, Davenport WD, Jeansonne BG. The effectof guided tissue regeneration on the healing of osseousdefects in rat calvaria. J Endod 1999;25:81-84.

35. Blom EJ, Klein-Nulend J, Yin L, van Waas MA, BurgerEH. Transforming growth factor-β1 incorporated in cal-cium phosphate cement stimulates osteotransductivityin rat calvarial bone defects. Clin Oral Implants Res2001;12:609-616.

36. Cacciafesta V, Dalstra M, Bosch C, Melsen B, AndreassenTT. Growth hormone treatment promotes guided boneregeneration in rat calvarial defects. Eur J Orthod2001;23:733-740.

37. Dupoirieux L, Pourquier D, Picot MC, Neves M. Com-parative study of three different membranes for guidedbone regeneration of rat cranial defects. Int J Oral Max-illofac Surg 2001;30:58-62.

38. Verna C, Bosch C, Dalstra M, Wikesjö UME, TrombelliL. Healing patterns in calvarial bone defects followingguided bone regeneration in rats: A micro-CT scananalysis. J Clin Periodontol 2002;29:865-870.

Correspondence: Dr. Molly E. Pryor, Laboratory for AppliedPeriodontal and Craniofacial Regeneration, Temple Univer-sity School of Dentistry, 3223 N. Broad St., Philadelphia, PA19140. E-mail: wikesjo@comcast.net.

Accepted for publication December 21, 2004.

40339.qxd 8/8/05 7:32 AM Page 1292