How to optimize the preparation of leukocyte- and platelet-rich fibrin (L-PRF, Choukroun's technique) clots and membranes: Introducing the PRF Box
2010; Elsevier BV; Volume: 110; Issue: 3 Linguagem: Inglês
10.1016/j.tripleo.2010.05.048
ISSN1528-395X
Autores Tópico(s)Oral and gingival health research
ResumoWe recently read a very interesting article by Su et al.1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar in OOOOE about the in vitro release of growth factors from a Choukroun's platelet-rich fibrin (PRF) clot after 5, 60, 120, and 300 minutes. The authors concluded that the initial release of the main growth factors was very important during the first minutes and therefore that the PRF membranes should be used as quickly as possible to get the best possible clinical results.That article confirmed our previously published data about the slow release of growth factors from a PRF membrane,2Dohan 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 where we quantified a significant release of growth factors during at least 7 days. However, the 2 series of data showed a different kinetics of slow release and led to slightly different conclusions concerning the best way to use PRF; it therefore requires some discussion.Different Methods, Different Results, One ConclusionWe have presented in the Table I the mean values of the slow release of 3 key growth factors quantified in both studies: platelet-derived growth factor (PDGF) AB, transforming growth factor (TGF) β1, and vascular endothelial growth factor (VEGF). The slow release of PDGF-AB and TGF-β1 was much slower in our study than in the Su et al. article. In contrast, the release of VEGF was much more important in our experiment. The comparison of these data should be interpreted with caution, because both methodologies are very different:Table ICumulative mean quantities of 3 growth factors (in nanograms) released by a platelet-rich fibrin (PRF) clot (Su et al., July 2009),1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar by a PRF membrane alone prepared with compresses (Dohan Ehrenfest et al., February 2009),2Dohan 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 and by a PRF membrane prepared with the PRF Box, during the first minutes and hours after preparation (note that the conditions of experimentation were different between our2Dohan 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 and Su et al.'s1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar studies)Growth factorPRF clot (membrane + exudate)1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google ScholarPRF membrane using compresses2Dohan 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 ScholarPRF membrane using the PRF Box5 min1 h5 h (300 min)20 min1 h4 h168 h20 min1 h4 h168 hPDGF-AB29.2934.1352.732.13.87.650.33.612.321.263.2TGF-β143.0649.5272.2116.327.544.3273.422.438.952.8295.1VEGF0.380.421.040.7981.6142.4026.0710.8811.7382.6036.362PDGF, Platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. Open table in a new tab In our study,2Dohan 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 the PRF initial serum exudate was gently flushed out on compresses and then the membrane alone was placed in a significant volume of culture medium (4 mL) to protect the cell integrity of the leukocytes living in the clot.3Dohan 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, 4Dohan Ehrenfest D.M. Diss A. Odin G. Doglioli P. Hippolyte M.P. Charrier J.B. In vitro effects of Choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 341-352Google Scholar, 5Dohan Ehrenfest D.M. Doglioli P. de Peppo G.M. Del Corso M. Charrier J.B. Choukroun's platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way.Arch Oral Biol. 2010; 55: 185-194Google Scholar The quantified growth factors were thus not only the content of the membrane, but also the factors massively produced by the leukocytes alive in the membrane. This explained a strong 7-day-long slow release of TGF-β1 and VEGF, massively produced by leukocytes.In the Su et al. study,1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar the complete PRF clots (membrane + initial serum exudate) were placed in a tube without additional culture medium, and intensively vortexed to flush out the exudate in the tube. The exudate was then tested at different times. With this methodology, growth factors were forcibly extracted from the PRF clot and the initial exudate was also analyzed; this method explains the very quick initial growth factor release. However, the viability of the leukocytes is uncertain with this method: these cells trapped in the fibrin matrix were probably damaged by the vortex-induced extraction and the osmotic shock (related to the absence of dilution in an isotonic solution or culture medium). This could explain the lower VEGF levels quantified by Su et al.The conclusions of Su et al. are thus debatable. In their study, the release kinetics of the growth factors was provoked by their method of extraction and analysis, and thus does not represent the physiological reality. We agree that significant amounts of growth factors are released during the first 20 minutes after preparation,2Dohan 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 and thus that PRF should be used as quickly as possible after preparation. However this release is considerably less significant when forcible extraction is avoided (as shown in our study). In physiologic conditions, the main release occurs after several hours,2Dohan 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 and PRF can be used a long time after preparation, as long as the material is conserved in the adequate conditions.Finally, Su et al. also performed a very interesting gel electrophoresis, and hypothesized that fibronectin was absent from the exudate and therefore trapped in the PRF clot architecture. This suggestion should, however, be reevaluated carefully. Indeed, we quantified recently with enzyme-linked immunosorbent assays the slow release of fibronectin from a PRF clot over 7 days, and this release kinetics was very similar to the profile of the 3 other growth factors. The initial exudate contained 3.1 μg fibronectin per clot, and 1 membrane released a mean 3.5 μg after 20 minutes, 5.3 μg after 1 hour, and 48.4 μg of fibronectin after 7 days (cumulative values). The released quantities were thus very important.Another interesting observation is that the released amount of PDGF-AB after 5 hours in Su et al.'s study was much larger (>6×) than the amount after 4 hours in our study, and just about equal to our amount after 7 days. The comparison of these data highlights that the initial exudate of the clot may be rich in growth factors and therefore that the way to flush this exudate out may be improved to stabilize larger quantities of growth factors within the membrane. Indeed, when preparing membranes with compresses, the initial release of PDGF-AB (and of TGF-β1 also, when considering the very first hours) seemed to be lost, even if the membrane remained very rich in growth factors and able to produce large amounts for several days. This observation also raised the question of the adequate way to conserve the PRF membrane when prepared, because keeping the membranes on compresses (even wet compresses) for too long would increase an untimely release of growth factors and the shrinkage of the fibrin network and could also dehydrate and damage the leukocyte content.This issue is particularly significant when considering that many clinicians using PRF do not understand that PRF is a living biomaterial3Dohan 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, 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 I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar, 7Dohan 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 that requires a good knowledge of the way to produce, prepare, and conserve it before use. The lack of knowledge leads to many mistakes in the preparation of PRF membranes, which finally leads to a damaged dry product and contradictory clinical results.8Del Corso M. Sammartino G. Dohan Ehrenfest D.M. Choukroun's platelet-rich fibrin (PRF) membranes in periodontal surgery: understanding the biomaterial or believing in the magic of growth factors?.J Periodontol. 2009; 80: 1694-1697Google Scholar We already noticed these issues in 2006, when we investigated the growth factor content of the exudate separately.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 II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar, 10Dohan 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 Based on these observations, the preparation and conservation methods have been improved by the development of alternate methods.From the Metal Spoon to the PRF BoxSu et al. proposed different options to improve the preparation protocol for PRF, such as the production of the PRF immediately before use and the use of a nonabsorbable impermeable sterile material and a sterile cuvette to squeeze the PRF clot and to recover this initial releasate rich in growth factors. The authors are on the right track, but these solutions have already existed for several years. Since 2005, we have thoroughly investigated different options to improve the conservation and preparation of PRF.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 II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar, 10Dohan 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 ScholarA first solution was to conserve the PRF clot in its centrifugation tube: as long as the serum has not been flushed away from the clot, the growth factor content remains stable. It is a good way to gain 5-15 minutes, but this is not a long-term solution, because the clot slowly starts to sink into the tube after centrifugation and to merge with the red blood cell base, leading to an unusable material loaded with red blood cells and with weak mechanical properties.A second solution was to keep the clots in a sterile metal cup and to press them into membranes with a sterile metal spoon when needed.3Dohan 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 The PRF initial exudate (rich in growth factors and serum proteins) is thus collected in the cup (and eventually usable for the hydratation of a bone biomaterial before grafting), and the PRF membranes are thus preserved in a wet serum environment. This method is effective from a biologic point of view, but it is not easy to handle when 8 or 16 PRF clots are produced.In 2007, a new device for the preparation and standardization of leukocyte- and platelet-rich fibrin (L-PRF) clots and membranes was invented by Dr. Joseph Choukroun: the PRF Box (Process, Nice, France). This metallic box was designed to collect and transform up to 16 PRF clots into membranes in sterile conditions at 1 time (Fig. 1) and to conserve them in a clean and wet environment before use. The Box also contains compression wells and maces to compress the PRF clots into dense PRF cylinders (Fig. 2), easy to use for filling cavities (such as extraction sockets). The serum exudate is collected in the second level of the box, can be used for a longer conservation of the membranes, and is ready to be mixed with a bone biomaterial for grafting. But this standardized approach also allows an increase in the total growth factors release of the PRF membrane itself.Fig. 2The PRF Box is a polyvalent tool. PRF clots (A) can be transformed into membranes (B), but also placed into plastic wells and compacted using a metal mace (arrows). C, The PRF cylinders collected in the compression wells are dense fibrin plugs easy to handle in oral surgery.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using exactly the same methods than in our first study,2Dohan 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 we analyzed the slow release of growth factors from membranes produced with the PRF Box from the same 4 healthy volunteer donors. The results are reported in the Table I. When membranes were prepared with the PRF Box, the mean PDGF-AB amounts were significantly higher (analysis of variance [ANOVA]: P < .01) at each experimental time and the mean TGF-β1 and VEGF amounts were significantly higher (P < .01) during the first 4 hours only than the respective growth factor amounts in membranes prepared with compresses.The explanation of this result is quite simple: using the PRF Box, the compression procedure of the clots into membranes is performed with a gentle, slow, and homogeneous pressure, and the final membrane always remains homogeneously wet and soaked with serum. This gentle method thus avoids extracting and losing a significant amount of extrinsic-incorporated platelet growth factors9Dohan 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; it is particularly obvious with PDGF-AB, because that growth factor is released only by platelets. In contrast, this method probably does not influence the intrinsic-incorporated growth factors, which are slowly released in very high amounts for several days in both methods. Moreover, on a longer-term basis (after 24 and 168 hours), the global released amounts of VEGF and TGF-β1 were not significantly different between the 2 methods of preparation, because these factors are massively produced by the leukocytes. Thus, the compress method also allows the preparation of a serum-rich membrane with living leukocytes, but it requires a more expert handling.8Del Corso M. Sammartino G. Dohan Ehrenfest D.M. Choukroun's platelet-rich fibrin (PRF) membranes in periodontal surgery: understanding the biomaterial or believing in the magic of growth factors?.J Periodontol. 2009; 80: 1694-1697Google Scholar, 11Mazor 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 ScholarIn conclusion, we want to thank Su et al. for their interesting work, which confirmed our own results and opened this important discussion about the best preparation procedure for PRF. In contrast to Su et al., our own data supported that a PRF membrane remains usable many hours after preparation, as long as the PRF is prepared correctly and conserved in physiologic conditions. Moreover, the use of the PRF Box, a user-friendly and inexpensive tool, allows to guarantee the adequate preparation of homogeneous PRF membranes with a higher growth factor content, to avoid the dehydratation or death of the leukocytes living in the PRF clot, and also to prevent the shrinkage of the fibrin matrix architecture. We believe that this tool is now the gold standard for Choukroun's PRF preparation. We recently read a very interesting article by Su et al.1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar in OOOOE about the in vitro release of growth factors from a Choukroun's platelet-rich fibrin (PRF) clot after 5, 60, 120, and 300 minutes. The authors concluded that the initial release of the main growth factors was very important during the first minutes and therefore that the PRF membranes should be used as quickly as possible to get the best possible clinical results. That article confirmed our previously published data about the slow release of growth factors from a PRF membrane,2Dohan 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 where we quantified a significant release of growth factors during at least 7 days. However, the 2 series of data showed a different kinetics of slow release and led to slightly different conclusions concerning the best way to use PRF; it therefore requires some discussion. Different Methods, Different Results, One ConclusionWe have presented in the Table I the mean values of the slow release of 3 key growth factors quantified in both studies: platelet-derived growth factor (PDGF) AB, transforming growth factor (TGF) β1, and vascular endothelial growth factor (VEGF). The slow release of PDGF-AB and TGF-β1 was much slower in our study than in the Su et al. article. In contrast, the release of VEGF was much more important in our experiment. The comparison of these data should be interpreted with caution, because both methodologies are very different:Table ICumulative mean quantities of 3 growth factors (in nanograms) released by a platelet-rich fibrin (PRF) clot (Su et al., July 2009),1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar by a PRF membrane alone prepared with compresses (Dohan Ehrenfest et al., February 2009),2Dohan 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 and by a PRF membrane prepared with the PRF Box, during the first minutes and hours after preparation (note that the conditions of experimentation were different between our2Dohan 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 and Su et al.'s1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar studies)Growth factorPRF clot (membrane + exudate)1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google ScholarPRF membrane using compresses2Dohan 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 ScholarPRF membrane using the PRF Box5 min1 h5 h (300 min)20 min1 h4 h168 h20 min1 h4 h168 hPDGF-AB29.2934.1352.732.13.87.650.33.612.321.263.2TGF-β143.0649.5272.2116.327.544.3273.422.438.952.8295.1VEGF0.380.421.040.7981.6142.4026.0710.8811.7382.6036.362PDGF, Platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. Open table in a new tab In our study,2Dohan 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 the PRF initial serum exudate was gently flushed out on compresses and then the membrane alone was placed in a significant volume of culture medium (4 mL) to protect the cell integrity of the leukocytes living in the clot.3Dohan 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, 4Dohan Ehrenfest D.M. Diss A. Odin G. Doglioli P. Hippolyte M.P. Charrier J.B. In vitro effects of Choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 341-352Google Scholar, 5Dohan Ehrenfest D.M. Doglioli P. de Peppo G.M. Del Corso M. Charrier J.B. Choukroun's platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way.Arch Oral Biol. 2010; 55: 185-194Google Scholar The quantified growth factors were thus not only the content of the membrane, but also the factors massively produced by the leukocytes alive in the membrane. This explained a strong 7-day-long slow release of TGF-β1 and VEGF, massively produced by leukocytes.In the Su et al. study,1Su C.Y. Kuo Y.P. Tseng Y.H. Su C.H. Burnouf T. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 56-61Google Scholar the complete PRF clots (membrane + initial serum exudate) were placed in a tube without additional culture medium, and intensively vortexed to flush out the exudate in the tube. The exudate was then tested at different times. With this methodology, growth factors were forcibly extracted from the PRF clot and the initial exudate was also analyzed; this method explains the very quick initial growth factor release. However, the viability of the leukocytes is uncertain with this method: these cells trapped in the fibrin matrix were probably damaged by the vortex-induced extraction and the osmotic shock (related to the absence of dilution in an isotonic solution or culture medium). This could explain the lower VEGF levels quantified by Su et al.The conclusions of Su et al. are thus debatable. In their study, the release kinetics of the growth factors was provoked by their method of extraction and analysis, and thus does not represent the physiological reality. We agree that significant amounts of growth factors are released during the first 20 minutes after preparation,2Dohan 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 and thus that PRF should be used as quickly as possible after preparation. However this release is considerably less significant when forcible extraction is avoided (as shown in our study). In physiologic conditions, the main release occurs after several hours,2Dohan 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 and PRF can be used a long time after preparation, as long as the material is conserved in the adequate conditions.Finally, Su et al. also performed a very interesting gel electrophoresis, and hypothesized that fibronectin was absent from the exudate and therefore trapped in the PRF clot architecture. This suggestion should, however, be reevaluated carefully. Indeed, we quantified recently with enzyme-linked immunosorbent assays the slow release of fibronectin from a PRF clot over 7 days, and this release kinetics was very similar to the profile of the 3 other growth factors. The initial exudate contained 3.1 μg fibronectin per clot, and 1 membrane released a mean 3.5 μg after 20 minutes, 5.3 μg after 1 hour, and 48.4 μg of fibronectin after 7 days (cumulative values). The released quantities were thus very important.Another interesting observation is that the released amount of PDGF-AB after 5 hours in Su et al.'s study was much larger (>6×) than the amount after 4 hours in our study, and just about equal to our amount after 7 days. The comparison of these data highlights that the initial exudate of the clot may be rich in growth factors and therefore that the way to flush this exudate out may be improved to stabilize larger quantities of growth factors within the membrane. Indeed, when preparing membranes with compresses, the initial release of PDGF-AB (and of TGF-β1 also, when considering the very first hours) seemed to be lost, even if the membrane remained very rich in growth factors and able to produce large amounts for several days. This observation also raised the question of the adequate way to conserve the PRF membrane when prepared, because keeping the membranes on compresses (even wet compresses) for too long would increase an untimely release of growth factors and the shrinkage of the fibrin network and could also dehydrate and damage the leukocyte content.This issue is particularly significant when considering that many clinicians using PRF do not understand that PRF is a living biomaterial3Dohan 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, 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 I: technological concepts and evolution.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e37-e44Google Scholar, 7Dohan 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 that requires a good knowledge of the way to produce, prepare, and conserve it before use. The lack of knowledge leads to many mistakes in the preparation of PRF membranes, which finally leads to a damaged dry product and contradictory clinical results.8Del Corso M. Sammartino G. Dohan Ehrenfest D.M. Choukroun's platelet-rich fibrin (PRF) membranes in periodontal surgery: understanding the biomaterial or believing in the magic of growth factors?.J Periodontol. 2009; 80: 1694-1697Google Scholar We already noticed these issues in 2006, when we investigated the growth factor content of the exudate separately.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 II: platelet-related biologic features.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 101: e45-e50Google Scholar, 10Dohan 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 Based on these observations, the preparation and conservation methods have been improved by the development of alternate methods. We have presented in the Table I the mean values of the slow release of 3 key growth factors quantified in both studies: platelet-derived growth factor (PDGF) AB, transforming growth factor (TGF) β1, and vascular endothelial growth factor (VEGF). The slow release of PDGF-AB and TGF-β1 was much slower in our study than in the Su et al. article. In contrast, the release of VEGF was much more important in our experiment. The comparison of these data should be interpreted with caution, because both methodologies are very different:
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