Selecting a relevant in vitro cell model for testing and comparing the effects of a Choukroun platelet-rich fibrin (PRF) membrane and a platelet-rich plasma (PRP) gel: Tricks and traps

Abstract

We have carefully read the letter by David M. Dohan Ehrenfest et al. regarding our article “A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro.” We respect Dr. Dohan Ehrenfest's pioneer work in PRF research. The purpose of our article was to evaluate the releasing pattern of growth factors in PRP and PRF and their effects on osteoblastic proliferation and differentiation. Our work was based on Dr. Dohan Ehrenfest's study that growth factors in PRF could release progressively and that this releasing pattern is thought to be one of the major differences between PRP and PRF in biologic activity.1Dohan 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 We would like to provide our detailed study materials and reply to Dr. Dohan Ehrenfest's questions as follows. In 2007, when we tried to study the releasing pattern of PRF, we did not read any study about growth factor–releasing patterns of PRF with exudate models, nor reports on its cell effects; therefore, we could not follow any reported protocol from the previous literature to design our work. In fact, our study showed the different releasing patterns of platelet-derived growth factor (PDGF) AB and transforming growth factor (TGF) β1 in exudates of PRF and PRP. Furthermore, we determined the different effects of exudates of PRP and PRF on osteoblastic proliferation and differentiation. The results of our study showed that exudates of PRF had a stronger and more durable effect on osteoblastic proliferation and differentiation than PRP. In 2009 and 2010, we read Dr. Dohan Ehrenfest's articles about osteoblast coculture with the whole PRF membrane for 7 days to assess osteoblastic proliferation and differentiation2Dohan 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, 3Dohan 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—after we had submitted our paper in March 2009. In our study, osteoblasts were stimulated continuously by exudates at different time points until the 28th day. Thus, releasing pattern and its effect on osteoblastic proliferation and differentiation of exudates containing growth factors at different time points (1, 7, 14, 21, and 28 days) were the main purposes of our study. The method used in our study is representative and typical in a certain way for testing the growth factor–releasing pattern of PRF. Our study reflected the significant difference of growth factor–releasing patterns and their cell effects between PRF and PRP through evaluation of osteoblastic properties stimulated by the exudates, which was thought to be one of major differences of PRP and PRF in their biologic properties. Our study was to test this one major difference, the complete function between PRP and PRF. PRP was prepared according to the Curasan protocol, and a standard laboratory centrifuge (Heraeus Labofuge 300; Kendro Laboratory Products, Osterrode, Germany) was used. Platelets were calculated before PRP was activated by the thrombin, as described in our article.4He L. Lin Y. Hu X. Zhang Y. Wu H. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 707-713Google Scholar PRF was prepared according to the standard protocol in Dr. Dohan Ehrenfest's study.5Dohan 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 Briefly, blood samples were taken without anticoagulant in 10-mL tubes (Vacutainer; BD, Franklin Lakes, NJ, USA) that were immediately centrifuged for 10 minutes at 400g in a standard laboratory centrifuge (Heraeus Labofuge 300), and a typical fibrin network of PRF as described before was produced (Fig. 1, Fig. 2, Fig. 3). Platelets in PRF were calculated using an indirect method, i.e., platelets in the whole blood were calculated first, then the platelet counts in the residual part after PRF preparation were subtracted.Fig. 2Preparation of PRF according to Dr. Dohan Ehrenfest's protocol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3The 3-dimensional structure of PRF under a scanning electron microscope.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The studies by Dohan Ehrenfest et al. reported that Curasan PRP and Choukroun PRF were respectively an L-PRP and an L-PRF and thus contained leukocytes, which might have a potential effect on the releasing of growth factors and a coculture effects with osteoblasts.2Dohan 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 In our study, we only observed the difference in releasing pattern of growth factors between PRF and PRP and in its further effects on rat osteoblasts. Therefore, we did not focus on evaluation of leukocyte effects separately. We agree with Dr. Dohan Ehrenfest's opinion on the methodology for testing the cell effects of PRP or PRF in human beings. It is reasonable to use human cells in primary cultures from the same donor as for the platelet concentrate to avoid immune cross-reactions and other related bias. However, in our study to avoid sample bias, we collected 14 blood samples from 14 human beings, but it is very difficult to obtain 14 bone samples from the same donor group in China. Rat osteoblasts were already used as cell model to test cell effects of PRP in earlier studies.6Soffer E. Ouhayoun J.P. Dosquet C. Meunier A. Anagnostou F. Effects of platelet lysates on select bone cell functions.Clin Oral Implants Res. 2004; 15: 581-588Google Scholar The results of our article also showed that rat osteoblasts proliferated and differentiated as well as the positive control group.4He L. Lin Y. Hu X. Zhang Y. Wu H. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108: 707-713Google Scholar The results implied that the rat osteoblast model for testing the cell effects of PRP and PRF was acceptable. However, in vitro results cannot fully reflect the in vivo situation. In our whole study, we prepared the PRF according to Dr. Dohan Ehrenfest's protocol (Fig. 1, Fig. 2, Fig. 3), but in writing the article I gave the wrong protocol name with “PCCS”; no deliberate intent was involved. This was a terrible mistake made by me. I deeply apologize to everyone for confusion or inconvenience caused by my mistake. I deeply apologize to Dr. Dohan for the unhappy or uncomfortable feelings caused by my mistake. I respect the pioneer work that Dr. Dohan Ehrenfest and his team did. This is really a lesson for me and I will be exceptionally cautious about such matters in the future. Furthermore, as a young postgraduate student with my first published paper, I really thank Dr. Dohan Ehrenfest for providing me such an opportunity to correct my mistakes and allow me to clarify this fundamental point. I respect Dr. Dohan Ehrenfest even more than before for his serious scientific spirit. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part III: Leucocyte activation: A new feature for platelet concentrates?Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and EndodonticsVol. 101Issue 3PreviewPlatelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this third article, we investigate the immune features of this biomaterial. During PRF processing, leucocytes could also secrete cytokines in reaction to the hemostatic and inflammatory phenomena artificially induced in the centrifuged tube. We therefore undertook to quantify 5 significant cell mediators within platelet poor plasma supernatant and PRF clot exudate serum: 3 proinflammatory cytokines (IL-1β, IL-6, and TNF-α), an antiinflammatory cytokine (IL-4), and a key growth promoter of angiogenesis (VEGF). Full-Text PDF Selecting a relevant in vitro cell model for testing and comparing the effects of a Choukroun's platelet-rich fibrin (PRF) membrane and a platelet-rich plasma (PRP) gel: Tricks and trapsOral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and EndodonticsVol. 110Issue 4PreviewWe recently read with a great interest an article published in OOOOE, where He et al.1 tried to compare the effects in vitro on the proliferation and differentiation of rat osteoblasts induced by a platelet-rich plasma (PR) and a platelet-rich fibrin (PRF). We have a significant experience with cell cultures using PRF,2,3 and the method and terminology used by He et al. raise several questions that require discussion. Full-Text PDF