Selecting a relevant animal model for testing the in vivo effects of Choukroun's platelet-rich fibrin (PRF): Rabbit tricks and traps

Abstract

We recently read 2 interesting articles1Lee E.H. Kim J.Y. Kweon H.Y. Jo Y.Y. Min S.K. Park Y.W. et al.A combination graft of low-molecular-weight silk fibroin with Choukroun's platelet-rich fibrin for rabbit calvarial defect.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: e33-e38Google Scholar, 2Jang E.S. Park J.W. Kweon H. Lee K.G. Kang S.W. Baek D.H. et al.Restoration of peri-implant defects in immediate implant installations by Choukroun's platelet-rich fibrin and silk fibroin powder combination graft.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: 831-836Google Scholar from the same team published in OOOOE about the use of a combination of Choukroun's platelet-rich fibrin (PRF) and silk fibroin as filling material for bone regeneration. Both studies were performed in a rabbit model: Lee et al.1Lee E.H. Kim J.Y. Kweon H.Y. Jo Y.Y. Min S.K. Park Y.W. et al.A combination graft of low-molecular-weight silk fibroin with Choukroun's platelet-rich fibrin for rabbit calvarial defect.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: e33-e38Google Scholar in bone calvarial defects and Jang et al.2Jang E.S. Park J.W. Kweon H. Lee K.G. Kang S.W. Baek D.H. et al.Restoration of peri-implant defects in immediate implant installations by Choukroun's platelet-rich fibrin and silk fibroin powder combination graft.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: 831-836Google Scholar in bone peri-implant defects (in the tibia). These studies are of great interest and open an important discussion about the best animal model to use when testing a product such as PRF.In both studies, the authors concluded that results from rabbit studies can not be directly extrapolated to human applications and therefore require further validation. We agree with the authors that all animal model results have to be interpreted carefully. It is particularly true when testing products such as PRF,3Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar which is highly dependent on the blood composition and coagulation mechanisms related to each species. But in this case, the main issue is much more practical: From our experience, it is impossible to produce true Choukroun's PRF in rabbits.Choukroun's PRF is a complex fibrin-based solid biomaterial4Dohan Ehrenfest D.M. Rasmusson L. Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).Trends Biotechnol. 2009; 27: 158-167Google Scholar presenting a specific cell content (leukocytes, platelet aggregates) and distribution,5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar matrix architecture,6Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar and growth factor release profile.7Dohan Ehrenfest D.M. de Peppo G.M. Doglioli P. Sammartino G. Slow release of growth factors and thrombospondin-1 in Choukroun's platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies.Growth Factors. 2009; 27: 63-69Google Scholar This architecture is highly stable as long as the main preparation rules are strictly followed.5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar One of the key principles is that blood must be collected directly in the preparation tube and immediately centrifuged. When the time for blood collection is too long and not homogeneous, blood starts to partially coagulate in the tube before centrifugation, and the whole harvesting should be discarded, because it will be impossible to get a well structured PRF clot. As shown in Fig. 1, an inadequate and slow blood harvesting and preparation process in human patients leads to a small PRF-like fibrin bulk, with unstable fibrin polymerization (resulting in weak mechanical properties) and an unknown and unreproducible growth factor and cell content. Moreover, it is very difficult to separate this small fibrin bulk from the red blood cell (RBC) base, resulting in a heavy load of RBCs within the product.As the first team publishing on Choukroun's PRF technology,3Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar, 6Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar, 8Choukroun J. Adda F. Schoeffler C. Vervelle A. Une opportunité en paro-implantologie: le PRF.Implantodontie. 2001; 42: 55-62Google Scholar, 9Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part III: leucocyte activation: a new feature for platelet concentrates?.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e51-e55Google Scholar, 10Choukroun J. Diss A. Simonpieri A. Girard M.O. Schoeffler C. Dohan S.L. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part IV: clinical effects on tissue healing.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e56-e60Google Scholar, 11Choukroun J. Diss A. Simonpieri A. Girard M.O. Schoeffler C. Dohan S.L. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: 299-303Google Scholar we have already tested this biomaterial in several animal models. We performed several studies in rabbits but never published those data, because it became obvious that the clots obtained in rabbits were not Choukroun's PRF. The reason is very simple: rabbits are small animals, with small veins and arteries,12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar and it is almost impossible to perform the homogeneous and quick collection of a significant volume of blood in a tube designed for PRF. And even with luck, when it works with one rabbit, it is impossible to reproduce the same adequate harvesting in a complete experimental series of rabbits.In their studies, Jang et al. and Lee et al. used animals with an average weight of 2.3-2.7 kg, i.e., quite small New Zealand white rabbits. They have long ears, and blood collection from the ear vein is possible.12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar However, the probability to harvest 10 mL from these veins in 1 quick and direct shot is nil. We have extensive experience in trying such collection, even after requiring the help of many well trained anesthesiologists (veterinary and human). We tried the procedure again with the biggest possible rabbits available (>5 kg), with smaller collection tubes (4.5 mL instead of 9 mL), and in many different veins or even arteries (marginal ear veins, central ear artery, jugular vein, cephalic vein, lateral saphenous veins). But even in the best possible conditions, the quick and direct collection remained uncertain. The rabbit skin and vessels are quite fragile (particularly when considering the size of our collection needles), vessels are not easily visualized (particularly in a fat 5-kg rabbit), and if we do not succeed in the first try, hematoma formation, bruising, vessel thrombosis, and skin/vessel sloughing quickly result.12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar The only technique that has allowed us to gather quickly a full tube (4.5 or 9 mL) was intracardiac harvesting, but this method led to a high mortality rate, even with an expert anesthesiologist harvesting.To have a quick blood collection, we first need good blood pressure and the associated blood volume. This is the main reason why rabbit is not an adequate model for PRF. Choukroun's PRF is a technique designed for humans, and therefore adapted to human blood pressure and volume. It is very easy to handle in all human-scaled (such as beagle dogs, baboons, and pigs) or larger (such as horses, goats, and cows) animals. As shown in Table I, each blood harvesting can severely perturbe the blood composition of the animals. In all species, the total collected blood volume should never exceed 15%-20%, or it can lead to a life-threatening situation for the animal. The collection of only 10% of the total blood volume requires 2 weeks of recovery for the restoration of blood normal composition, and 15% requires 4 weeks of recovery. In a big 4-kg rabbit, this 10% limit is ∼22 mL blood. In a 2.3-kg rabbit, the blood volume collected by Lee et al. was very close to this 10% limit. In fact, even with very big rabbits, the volume of blood that can be drawn without creating a severe decrease of the circulating leukocytes and platelets is very small. In contrast, producing larger volumes of PRF is not a problem with bigger animals, such as dogs or pigs. Unfortunately, we know many researchers who have tried to produce PRF in rabbits and did not realize that this model was intrinsically wrong, even though they all concede that a quick blood collection in rabbits is difficult.Table IMean blood volumes of a few species commonly used in animal experimentsSpecies (wt)Total blood volume (mL)10% of the total blood volume (mL)Mouse (25 g)1.80.2Rat (250 g)161.6Rabbit (4 kg)22022Dog (10 kg)85085Pig (30 kg)1,950195The collection of 10% of the total blood volume typically requires 2 weeks' recovery before restoration of the normal blood composition (15% requires 4 weeks' recovery). Open table in a new tab The question, therefore, is to understand what kind of product Lee et al. and Jang et al. tested in their articles, because it was obviously not true Choukroun's PRF. In both articles, 10-mL blood samples were taken from the ear vein of each rabbit and then centrifuged. Jang et al. showed the collection and preparation procedure in 2 photos, and a striking detail can be observed: Blood was collected using a plastic syringe (aspirating blood forcibly in the ear vein) and then transfered into an Eppendorf-like tube (probably a 2-mL tube) for centrifugation. This method in 2 steps is critical, because it takes a lot of time to fill the syringe, and even more time to fill the tubes for centrifugation. Moreover, because blood starts to coagulate in the syringe, we have no idea of the content of the blood sample transfered into the tube and processed into a PRF-like structure. Last but not least, the authors did not use the original glass tubes or glass-coated plastic tubes,5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar but a simple Eppendorf plastic tube; in our experience, these kinds of plastic tubes lead to an incoherent fibrin bulk. For all of these reasons, the method used by these authors for the production of a PRF clot was inadequate and led to the production of a small fibrin bulk with an unknown cell and growth factor content and an unstable fibrin architecture. In short, this was not Choukroun's PRF.Finally, in this small animal model, the final product was barely usable. Lee et al. indeed complained that the volume of fibrin bulk was limited because of the limited sampling size, and they concluded that when the defect size is large, Choukroun's PRF has to be used in combination with other filling materials. As already proven in sinus-lift procedures13Mazor Z. Horowitz R.A. Del Corso M. Prasad H.S. Rohrer M.D. Dohan Ehrenfest D.M. Sinus floor augmentation with simultaneous implant placement using Choukroun's platelet-rich fibrin as the sole grafting material: a radiologic and histologic study at 6 months.J Periodontol. 2009; 80: 2056-2064Google Scholar and in plastic surgery,14Charrier J.B. Monteil J.P. Albert S. Collon S. Bobin S. Dohan Ehrenfest D.M. Relevance of Choukroun's platelet-rich fibrin (PRF) and SMAS flap in primary reconstruction after superficial or subtotal parotidectomy in patients with focal pleiomorphic adenoma: a new technique.Rev Laryngol Otol Rhinol (Bord). 2008; 129: 313-318Google Scholar, 15Braccini F. Dohan D.M. [Relevance of Choukroun's platelet-rich fibrin (PRF) during facial esthetic lipostructure (Coleman technique): preliminary results].Rev Laryngol Otol Rhinol (Bord). 2007; 128 (French): 255-260Google Scholar this statement is not true in humans. PRF clots, when produced correctly and in the adequate species, represent a significant filling volume.In conclusion, Choukroun's PRF has been designed by a human anesthesiologist8Choukroun J. Adda F. Schoeffler C. Vervelle A. Une opportunité en paro-implantologie: le PRF.Implantodontie. 2001; 42: 55-62Google Scholar and requires a minimum blood pressure and volume to be adapted to other species. In their articles, Jang et al. and Lee et al. gave very interesting results, and their combination of PRF with silk fibroin is an interesting approach. Unfortunately, they did not use Choukroun's PRF in their studies, but only a poor-quality PRF-like fibrin bulk. We can only surmise that the results may have been better with a true PRF clot. We hope that in future studies of PRF, colleagues will avoid the rabbit model and use larger animals, such as dogs or goats. We recently read 2 interesting articles1Lee E.H. Kim J.Y. Kweon H.Y. Jo Y.Y. Min S.K. Park Y.W. et al.A combination graft of low-molecular-weight silk fibroin with Choukroun's platelet-rich fibrin for rabbit calvarial defect.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: e33-e38Google Scholar, 2Jang E.S. Park J.W. Kweon H. Lee K.G. Kang S.W. Baek D.H. et al.Restoration of peri-implant defects in immediate implant installations by Choukroun's platelet-rich fibrin and silk fibroin powder combination graft.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: 831-836Google Scholar from the same team published in OOOOE about the use of a combination of Choukroun's platelet-rich fibrin (PRF) and silk fibroin as filling material for bone regeneration. Both studies were performed in a rabbit model: Lee et al.1Lee E.H. Kim J.Y. Kweon H.Y. Jo Y.Y. Min S.K. Park Y.W. et al.A combination graft of low-molecular-weight silk fibroin with Choukroun's platelet-rich fibrin for rabbit calvarial defect.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: e33-e38Google Scholar in bone calvarial defects and Jang et al.2Jang E.S. Park J.W. Kweon H. Lee K.G. Kang S.W. Baek D.H. et al.Restoration of peri-implant defects in immediate implant installations by Choukroun's platelet-rich fibrin and silk fibroin powder combination graft.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109: 831-836Google Scholar in bone peri-implant defects (in the tibia). These studies are of great interest and open an important discussion about the best animal model to use when testing a product such as PRF. In both studies, the authors concluded that results from rabbit studies can not be directly extrapolated to human applications and therefore require further validation. We agree with the authors that all animal model results have to be interpreted carefully. It is particularly true when testing products such as PRF,3Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar which is highly dependent on the blood composition and coagulation mechanisms related to each species. But in this case, the main issue is much more practical: From our experience, it is impossible to produce true Choukroun's PRF in rabbits. Choukroun's PRF is a complex fibrin-based solid biomaterial4Dohan Ehrenfest D.M. Rasmusson L. Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).Trends Biotechnol. 2009; 27: 158-167Google Scholar presenting a specific cell content (leukocytes, platelet aggregates) and distribution,5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar matrix architecture,6Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar and growth factor release profile.7Dohan Ehrenfest D.M. de Peppo G.M. Doglioli P. Sammartino G. Slow release of growth factors and thrombospondin-1 in Choukroun's platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies.Growth Factors. 2009; 27: 63-69Google Scholar This architecture is highly stable as long as the main preparation rules are strictly followed.5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar One of the key principles is that blood must be collected directly in the preparation tube and immediately centrifuged. When the time for blood collection is too long and not homogeneous, blood starts to partially coagulate in the tube before centrifugation, and the whole harvesting should be discarded, because it will be impossible to get a well structured PRF clot. As shown in Fig. 1, an inadequate and slow blood harvesting and preparation process in human patients leads to a small PRF-like fibrin bulk, with unstable fibrin polymerization (resulting in weak mechanical properties) and an unknown and unreproducible growth factor and cell content. Moreover, it is very difficult to separate this small fibrin bulk from the red blood cell (RBC) base, resulting in a heavy load of RBCs within the product. As the first team publishing on Choukroun's PRF technology,3Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar, 6Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar, 8Choukroun J. Adda F. Schoeffler C. Vervelle A. Une opportunité en paro-implantologie: le PRF.Implantodontie. 2001; 42: 55-62Google Scholar, 9Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part III: leucocyte activation: a new feature for platelet concentrates?.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e51-e55Google Scholar, 10Choukroun J. Diss A. Simonpieri A. Girard M.O. Schoeffler C. Dohan S.L. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part IV: clinical effects on tissue healing.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e56-e60Google Scholar, 11Choukroun J. Diss A. Simonpieri A. Girard M.O. Schoeffler C. Dohan S.L. et al.Platelet-rich fibrin (PRF): a second-generation platelet concentrate Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: 299-303Google Scholar we have already tested this biomaterial in several animal models. We performed several studies in rabbits but never published those data, because it became obvious that the clots obtained in rabbits were not Choukroun's PRF. The reason is very simple: rabbits are small animals, with small veins and arteries,12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar and it is almost impossible to perform the homogeneous and quick collection of a significant volume of blood in a tube designed for PRF. And even with luck, when it works with one rabbit, it is impossible to reproduce the same adequate harvesting in a complete experimental series of rabbits. In their studies, Jang et al. and Lee et al. used animals with an average weight of 2.3-2.7 kg, i.e., quite small New Zealand white rabbits. They have long ears, and blood collection from the ear vein is possible.12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar However, the probability to harvest 10 mL from these veins in 1 quick and direct shot is nil. We have extensive experience in trying such collection, even after requiring the help of many well trained anesthesiologists (veterinary and human). We tried the procedure again with the biggest possible rabbits available (>5 kg), with smaller collection tubes (4.5 mL instead of 9 mL), and in many different veins or even arteries (marginal ear veins, central ear artery, jugular vein, cephalic vein, lateral saphenous veins). But even in the best possible conditions, the quick and direct collection remained uncertain. The rabbit skin and vessels are quite fragile (particularly when considering the size of our collection needles), vessels are not easily visualized (particularly in a fat 5-kg rabbit), and if we do not succeed in the first try, hematoma formation, bruising, vessel thrombosis, and skin/vessel sloughing quickly result.12Graham J. Common procedures in rabbits.Vet Clin North Am Exot Anim Pract. 2006; 9 (vii): 367-388Google Scholar The only technique that has allowed us to gather quickly a full tube (4.5 or 9 mL) was intracardiac harvesting, but this method led to a high mortality rate, even with an expert anesthesiologist harvesting. To have a quick blood collection, we first need good blood pressure and the associated blood volume. This is the main reason why rabbit is not an adequate model for PRF. Choukroun's PRF is a technique designed for humans, and therefore adapted to human blood pressure and volume. It is very easy to handle in all human-scaled (such as beagle dogs, baboons, and pigs) or larger (such as horses, goats, and cows) animals. As shown in Table I, each blood harvesting can severely perturbe the blood composition of the animals. In all species, the total collected blood volume should never exceed 15%-20%, or it can lead to a life-threatening situation for the animal. The collection of only 10% of the total blood volume requires 2 weeks of recovery for the restoration of blood normal composition, and 15% requires 4 weeks of recovery. In a big 4-kg rabbit, this 10% limit is ∼22 mL blood. In a 2.3-kg rabbit, the blood volume collected by Lee et al. was very close to this 10% limit. In fact, even with very big rabbits, the volume of blood that can be drawn without creating a severe decrease of the circulating leukocytes and platelets is very small. In contrast, producing larger volumes of PRF is not a problem with bigger animals, such as dogs or pigs. Unfortunately, we know many researchers who have tried to produce PRF in rabbits and did not realize that this model was intrinsically wrong, even though they all concede that a quick blood collection in rabbits is difficult. The collection of 10% of the total blood volume typically requires 2 weeks' recovery before restoration of the normal blood composition (15% requires 4 weeks' recovery). The question, therefore, is to understand what kind of product Lee et al. and Jang et al. tested in their articles, because it was obviously not true Choukroun's PRF. In both articles, 10-mL blood samples were taken from the ear vein of each rabbit and then centrifuged. Jang et al. showed the collection and preparation procedure in 2 photos, and a striking detail can be observed: Blood was collected using a plastic syringe (aspirating blood forcibly in the ear vein) and then transfered into an Eppendorf-like tube (probably a 2-mL tube) for centrifugation. This method in 2 steps is critical, because it takes a lot of time to fill the syringe, and even more time to fill the tubes for centrifugation. Moreover, because blood starts to coagulate in the syringe, we have no idea of the content of the blood sample transfered into the tube and processed into a PRF-like structure. Last but not least, the authors did not use the original glass tubes or glass-coated plastic tubes,5Dohan Ehrenfest D.M. Del Corso M. Diss A. Mouhyi J. Charrier J.B. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.J Periodontol. 2010; 81: 546-555Google Scholar but a simple Eppendorf plastic tube; in our experience, these kinds of plastic tubes lead to an incoherent fibrin bulk. For all of these reasons, the method used by these authors for the production of a PRF clot was inadequate and led to the production of a small fibrin bulk with an unknown cell and growth factor content and an unstable fibrin architecture. In short, this was not Choukroun's PRF. Finally, in this small animal model, the final product was barely usable. Lee et al. indeed complained that the volume of fibrin bulk was limited because of the limited sampling size, and they concluded that when the defect size is large, Choukroun's PRF has to be used in combination with other filling materials. As already proven in sinus-lift procedures13Mazor Z. Horowitz R.A. Del Corso M. Prasad H.S. Rohrer M.D. Dohan Ehrenfest D.M. Sinus floor augmentation with simultaneous implant placement using Choukroun's platelet-rich fibrin as the sole grafting material: a radiologic and histologic study at 6 months.J Periodontol. 2009; 80: 2056-2064Google Scholar and in plastic surgery,14Charrier J.B. Monteil J.P. Albert S. Collon S. Bobin S. Dohan Ehrenfest D.M. Relevance of Choukroun's platelet-rich fibrin (PRF) and SMAS flap in primary reconstruction after superficial or subtotal parotidectomy in patients with focal pleiomorphic adenoma: a new technique.Rev Laryngol Otol Rhinol (Bord). 2008; 129: 313-318Google Scholar, 15Braccini F. Dohan D.M. [Relevance of Choukroun's platelet-rich fibrin (PRF) during facial esthetic lipostructure (Coleman technique): preliminary results].Rev Laryngol Otol Rhinol (Bord). 2007; 128 (French): 255-260Google Scholar this statement is not true in humans. PRF clots, when produced correctly and in the adequate species, represent a significant filling volume. In conclusion, Choukroun's PRF has been designed by a human anesthesiologist8Choukroun J. Adda F. Schoeffler C. Vervelle A. Une opportunité en paro-implantologie: le PRF.Implantodontie. 2001; 42: 55-62Google Scholar and requires a minimum blood pressure and volume to be adapted to other species. In their articles, Jang et al. and Lee et al. gave very interesting results, and their combination of PRF with silk fibroin is an interesting approach. Unfortunately, they did not use Choukroun's PRF in their studies, but only a poor-quality PRF-like fibrin bulk. We can only surmise that the results may have been better with a true PRF clot. We hope that in future studies of PRF, colleagues will avoid the rabbit model and use larger animals, such as dogs or goats. The route from mission impossible to Columbus's egg: An easy means of platelet-rich-fibrin (PRF) production in the rabbitOral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and EndodonticsVol. 110Issue 4PreviewThe authors raised an interesting and practical issue regarding the experimental animal. The authors claimed that in their experience it is impossible to produce true Choukroun's platelet-rich-plasma (PRF) in rabbits. The authors supposed that our Choukroun's PRF might not be true Choukroun's PRF because we did not use a glass tube. According to an earlier publication, Choukroun's PRF should be produced by a single centrifugation of sampled blood without an anticoagulant or bovine thrombin as an additive. Full-Text PDF