Cytotoxicity analyses of Choukroun’s platelet-rich fibrin (PRF) on a wide range of human cells: The answer to a commercial controversy

Abstract

Several important issues have been questioned following a reader’s mail concerning the type of tubes used to produce platelet-rich fibrin (PRF) and the possible cytotoxicity of silica-releasing tubes (glass-coated plastic tubes) for the recipient organism. In fact, numerous silica-based materials are used in dentistry, including as a direct bone-filling material. The present paper answers the questions step by step, then describes a cytotoxicity study carried on 4 different human cell types (gingival fibroblasts, keratinocytes, preadipocytes, and osteoblasts) placed in contact with PRF membranes for 12 hours, 24 hours, 3 days, and 7 days, respectively, of in vitro culture. Cell metabolic activity was evaluated with succinic dehydrogenase activity, which measures the mitochondrial respiration of cells. For this purpose, the methyltetrazolium (MTT) assay was used. The results show that PRF produced with glass-coated plastic tubes is not cytotoxic for these human cells and for some even seems to improve the mitochondrial respiration. Platelet-rich fibrin is a second-generation platelet concentrate which allows one to obtain, starting from an anticoagulant-free blood harvest, fibrin membranes enriched with platelets and growth factors. The PRF protocol was described for the first time in 2001 by Dr. Joseph Choukroun et al.1Choukroun J. Adda F. Schoeffler C. Vervelle A. Une opportunité en paro-implantologie: le PRF.Implantodontie. 2001; 42: 55-62Google Scholar The protocol is not linked to a medical device nor to a specific machine: It is a general protocol, a simplified technique, free and openly accessible for all clinicians. It is not a blood-derived product, in contrast to the platelet-rich plasmas (PRPs) and fibrin glues2Dohan D.M. Choukroun J. PRP, cPRP, PRF, PRG, PRGF, FC … how to find your way in the jungle of platelet concentrates?.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007; 103: 305-306Google Scholar; to produce PRF, blood composition is not modified by the use of bovine thrombin, anticoagulants, or calcium chloride. Polymerization of PRF is performed according to a completely natural process, without any modifiers. Therefore, it is a totally autologous material prepared extemporaneously, in the same way as a bone harvest (chin, retromandibular line, illiac, parietal) or a palatal connective tissue harvest. The name “PRF” is protected by a copyright to make sure that our research work on this free and open-access protocol is not distorted by commercial companies interested in using the name. Mr. O’Connell raises an interesting question concerning the type of tubes to be used preferentially to produce PRF. Indeed, in our first international paper on the topic,3Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. Gogly B. 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, 4Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. Gogly B. 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, 5Dohan D.M. Choukroun J. Diss A. Dohan S.L. Dohan A.J. Mouhyi J. Gogly B. Platelet-rich fibrin (PRF): a second-generation platelet concentrate.Part III: leucocyte activation: a new feature for platelet concentrates?. 2006; 101: e51-e55Google Scholar, 6Choukroun 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, 7Choukroun 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 presented the standard protocol performed in the French dental offices, i.e., using glass-coated plastic tubes. However, the picture illustrated in that paper displayed a dry glass tube. In fact, the technique works with any type of dry glass tube (Terumo® Venoject® 10 mL) or glass-coated plastic tube (Terumo® Venosafe® 10 mL, Becton Dickinson Vacutainer® 10 mL or Greiner® Vacuette® 9 mL). Platelet-rich fibrin was initially developed in dry glass tubes as early as 2000. In the medical field, the conventional PRF protocol has continued to be performed in these dry glass tubes, such as those available in the French hospitals. Most maxillofacial, ENT, and plastic surgeons, using PRF on a conventional basis, produce their PRF using a Coleman fat centrifuge adapted for this specific indication to standard glass collection tubes (Fig. 1). In a dental office, the problem is different. French dentists are not initially educated to perform blood harvests. Therefore, the use of plastic tubes has been recommended to avoid tube breaking and contaminations during these quite exceptional handlings in a dental office. However, with plastic tubes alone, PRF can not be obtained. The contact with silica is necessary to start the polymerization process: The silica behaves as a clot activator. To produce PRF, either dry glass tubes (because they obviously answer all the enforced standards) or glass-coated plastic tubes must be used. The commercially available tubes are generally for diagnostic use only. Therefore, the question is to know whether we are allowed to use such tubes to produce the PRF membranes. We shall therefore answer step by step the remarks questioned. 1. Do the tubes used to procedure PRF have to comply with the ISO 10993 standard? First of all, we must define the reference standard: Is it the ISO 10993-4 or the ISO 10993-5? The ISO 10993-4 (2002) provides general requirements for evaluating the interactions of medical devices with blood. A classification of medical and dental devices that are intended for use in contact with blood is described, based on the intended use and duration of contact as defined in ISO 10993-1. The basic principles which rule the evaluation of the interaction of devices with blood, the rationale for careful selection of tests according to specific categories, together with the principles and scientific basis of these tests, are defined. Detailed requirements for testing cannot be specified because of limitations in the knowledge and accuracy of tests for blood-interacting devices. In ISO 10993-4 (2002), the biologic evaluation was described in general terms, and therefore this standard may not necessarily provide sufficient guidance for testing methods for a specific device. If one wishes to use the glass-coated plastic tubes under this standard, the problem is quite simple: It is impossible. The potential sanitary risk just cannot be demonstrated according to this standard. If the harvesting tubes are used for “in vitro use only,” it is not because they are dangerous for human health, but just because the manufacturers did not anticipate the use of these tubes for something other than harvesting blood for analyses. Facing this problem of standards and classification, the simplest solution would be to use dry glass tubes; the ISO 10993 standard would then be necessarily respected, and the same PRF produced. However, may glass-coated plastic tubes be used without any sanitary or forensic risk? The answer to this dilemna may be found in the French regulations. French legislation does not consider PRF to be a transfusion technique for blood-derived products (such as fibrin glues and PRPs), but as an autologous tissue graft (Fig. 2). Therefore, the ISO 10993-4 standard does not have to be applied to the harvesting technique and the PRF production. For security matters, PRF is ranked as an autologous biomaterial, and so we performed, as early as 2000, the relevant cytotoxicity tests based on the 10993-5 standard (1999). This aspect of the standard deals with the biologic evaluation of medical devices and defines tests for in vitro cytotoxicity. However it is important to recall that this standard can not be applied adequately to dental biomaterials, because it is too restrictive.8Susini G. About I. Tran-Hung L. Camps J. Cytotoxicity of Epiphany and Resilon with a root model.Int Endod J. 2006; 39: 940-944Google Scholar Indeed, numerous biomaterials used in dentistry demonstrate a well established cytotoxicity to various degrees.9Vajrabhaya L.O. Suwannawong S.K. Kamolroongwarakul R. Pewklieng L. Cytotoxicity evaluation of gutta-percha solvents: chloroform and GP-solvent (limonene).Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004; 98: 756-759Google Scholar, 10Vajrabhaya L.O. Korsuwannawong S. Jantarat J. Korre S. Biocompatibility of furcal perforation repair material using cell culture technique: Ketac Molar versus ProRoot MTA.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 102: e48-e50Google Scholar, 11Souza N.J. Justo G.Z. Oliveira C.R. Haun M. Bincoletto C. Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte-macrophage progenitor cells.Int Endod J. 2006; 39: 40-47Google Scholar In summary, the ISO 10993 standard can not be adequately applied to PRF. 2. The Becton Dickinson technical file recalls that silica dust (aluminum silicate) is a recognized toxic agent but only when used at high concentrations and inhaled. It specifies as well that no primary irritant effect or sensitization due to silica has been established on the skin or the eyes. The toxic effects of silica (particularly phosphorus or aluminum silicate) have been observed for over a century in populations exposed to high concentrations of silica in the inhaled air (specifically for miners and in some industries).12Chen W. Hnizdo E. Chen J.Q. Attfield M.D. Gao P. Hearl F. et al.Risk of silicosis in cohorts of Chinese tin and tungsten miners, and pottery workers (I): an epidemiological study.Am J Ind Med. 2005; 48: 1-9Google Scholar, 13Harrison J. Chen J.Q. Miller W. Chen W. Hnizdo E. Lu J. et al.Risk of silicosis in cohorts of Chinese tin and tungsten miners and pottery workers (II): workplace-specific silica particle surface composition.Am J Ind Med. 2005; 48: 10-15Google Scholar, 14Maxim L.D. Hadley J.G. Potter R.M. Niebo R. The role of fiber durability/biopersistence of silica-based synthetic vitreous fibers and their influence on toxicology.Regul Toxicol Pharmacol. 2006; 46: 42-62Google Scholar The silica particles deposit heavily in the bronchi, inducing a severe intoxication of the weakest cells and significant cell death. Prolonged exposure leads to overloaded cancer-free lung diseases15Elmore A.R. Final report on the safety assessment of aluminum silicate, calcium silicate, magnesium aluminum silicate, magnesium silicate, magnesium trisilicate, sodium magnesium silicate, zirconium silicate, attapulgite, bentonite, Fuller’s earth, hectorite, kaolin, lithium magnesium silicate, lithium magnesium sodium silicate, montmorillonite, pyrophyllite, and zeolite.Int J Toxicol. 2003; 22: 37-102Google Scholar, 16Elmore A.R. Final report on the safety assessment of potassium silicate, sodium metasilicate, and sodium silicate.Int J Toxicol. 2005; 24: 103-117Google Scholar known as silicosis. It is a long-term chronic intoxication.17Hnizdo E. Vallyathan V. Chronic obstructive pulmonary disease due to occupational exposure to silica dust: a review of epidemiological and pathological evidence.Occup Environ Med. 2003; 60: 237-243Google Scholar, 18Hnizdo E. Murray J. Risk of pulmonary tuberculosis relative to silicosis and exposure to silica dust in South African gold miners.Occup Environ Med. 1998; 55: 496-502Google Scholar, 19Hnizdo E. Murray J. Klempman S. Lung cancer in relation to exposure to silica dust, silicosis and uranium production in South African gold miners.Thorax. 1997; 52: 271-275Google Scholar Very high levels of silica are required to induce a cell effect detectable in vitro.20Murphy E.J. Roberts E. Horrocks L.A. Aluminum silicate toxicity in cell cultures.Neuroscience. 1993; 55: 597-605Google Scholar In dentistry, silica-based materials are in current use, because they belong to the dental biomaterials that offer the best tolerance with regard to living tissues, specifically bone. It must be recalled that most dental biomaterials are self-allergizing and toxic (resin composites, cements, etc.).21Geurtsen W. Toxicology of dental materials and “clinical experience.”.J Dent Res. 2003; 82: 500Google Scholar, 22Geurtsen W. Spahl W. Muller K. Leyhausen G. Aqueous extracts from dentin adhesives contain cytotoxic chemicals.J Biomed Mater Res. 1999; 48: 772-777Google Scholar, 23Souza P.P. Aranha A.M. Hebling J. Giro E.M. Costa C.A. In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements.Dent Mater. 2006; 22: 838-844Google Scholar 3. Theoretically, patients treated with PRF could be placed in contact with silica particles. But this possible contamination by a few microparticles of silica powder does not provide any health hazard. In dental surgery, silica particles are very often placed in contact with living tissues: All of our ceramics and many of our cement materials (e.g., glass ionomer) contain high levels of silica.23Souza P.P. Aranha A.M. Hebling J. Giro E.M. Costa C.A. In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements.Dent Mater. 2006; 22: 838-844Google Scholar, 24Aranha A.M. Giro E.M. Souza P.P. Hebling J. de Souza Costa C.A. Effect of curing regime on the cytotoxicity of resin-modified glass-ionomer lining cements applied to an odontoblast-cell line.Dent Mater. 2006; 22: 864-869Google Scholar, 25Sipahi C. Ozen J. Ural A.U. Dalkiz M. Beydemir B. The effect of two fibre impregnation methods on the cytotoxicity of a glass and carbon fibre-reinforced acrylic resin denture base material on oral epithelial cells and fibroblasts.J Oral Rehabil. 2006; 33: 666-673Google Scholar Indeed, we have numerous situations in which silica is in direct contact with bone and blood cells. During apical resections, glass ionomer is currently used as a filling material.26Karimjee C.K. Koka S. Rallis D.M. Gound T.G. Cellular toxicity of mineral trioxide aggregate mixed with an alternative delivery vehicle.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 102: e115-e120Google Scholar We may also mention the vitroceramic implants: Their surface contains a high level of silica, which allows fibrin nucleation and consequently osseointegration of these implants. In addition, some bone-grafting materials are bioglasses: Novabone Putty BioGlass, PerioGlas, etc. These products are approved by the FDA and are supposed to induce bone stimulation via ionic exchanges with bone cells. They are partially composed of calcium–phosphorus–sodium silicate. According to the companies who commercialize these biomaterials, the phosphorylated silicas are supposed to accelerate bone regeneration. Finally, all glass tubes release silica. This is proved by the production of PRF using dry glass tubes: With dry plastic tubes, there is no fibrin clot and no PRF can be obtained. Therefore, the dry glass tube releases silica. This observation includes an obvious fact: all glass tubes used in medical devices (or simply to store drugs, such as perfusion phials or flasks) release a minimal quantity of silica, which is hardly detectable on the contacting walls. This small quantity is assimilated to a silica powder deposit which coats the plastic tubes mentioned in the paper. This is the reason why most manufacturers have replaced their dry glass tubes by glass-coated plastic tubes, which are equivalent from a biochemical point of view. Should we consider that all of the drugs in glass tubes are toxic or Mr. O’Connell the that when the PRF the harvesting tube is and therefore blood may be This a of knowledge of PRF is fibrin for use, and therefore a autologous in the same way as a bone or an tissue one must be careful to avoid but the are the same as for any of autologous during we consider a bone or a palatal connective tissue graft as an of the answer is In we would to Mr. O’Connell for these interesting technical issues in It allowed to provide we are to provide the cytotoxicity carried on PRF using 4 different cell following relevant to the ISO 10993-5 standard. To perform cell in the of PRF, we to harvest tissue from patients to blood collection for produce the PRF required for Indeed, for requirements of PRF membranes must from the same as the cells. For this the human performed on to All of a small tissue harvest during a A different was for tested cell To fibroblasts, a was on the To a bone harvest was To preadipocytes, mL of fat from the of the The harvest, in preadipocytes, allows one to fat cells for culture A.M. C. G. C. The human tissue is a of 2005; Scholar, A.M. C. F. J. C. P. et of cells established from human 2004; Scholar To keratinocytes, a of was in the of the The carried and in at then placed in culture according to the the the cell then at For tested cell culture cells 4 used as and 4 a PRF from from the same as the The PRF was produced according to the protocol described using glass-coated plastic tubes Dickinson 10 mL). a of was for the at 12 24 3 and 7 All of the cell performed at and of culture in to which at at and at The culture was or 3 days, according to the cell was to the of of cell a tissue culture for and for placed in the for 24 to a cell well was with solution and the The 3 to This assay on the of the mitochondrial in living cells to the This is in the of the living cells. The of is to the mitochondrial solution was to The was placed in a for 4 the the solution was from the and well was with was well to the in the of The on the for to the of The was at with a using as the Detailed for these have been described by assay for growth and to and cytotoxicity Scholar The of the was to a of the as of the was performed by of and in of significant the was used. was when This of cytotoxicity tests the of cytotoxicity of PRF (Fig. there is a significant the and the that the allows one to the mitochondrial we may consider that at cell and preadipocytes, placed in contact with PRF, than the cells. a PRF produced in glass-coated plastic tubes no cytotoxicity The even seems This commercial is of the biologic effects of dental materials is of because their effects may they W. Toxicology of dental materials and “clinical experience.”.J Dent Res. 2003; 82: 500Google Scholar The ISO 10993-5 standard is for testing biocompatibility of medical devices with no specific indication for dental devices. Therefore, are often for dental and to clinical be For PRF, only cytotoxicity study of the as a is the of a possible of PRF is not an protocol could have been the culture in contact with the PRF membranes during 12 hours, then testing the on a of human cells placed in a tissue culture We did not that protocol, because the membranes release many growth and therefore the possible cytotoxicity of silica (or of PRF would be totally by the effect of the For this same some other conventional tests and are not relevant to the cytotoxicity of PRF. the cells on PRF present no of The PRF technique was initially developed using dry glass tubes, for which the of cytotoxicity does not In the first international paper the PRF technique, we on the glass-coated plastic tubes. Indeed, the use of plastic tubes security in of in a dental office, glass tubes may to be dangerous in of and This in all because the manufacturers only plastic tubes In the silica microparticles these tubes a quite risk of in contrast to bovine to and fibrin R.M. Platelet-rich for bone Surg Oral Med Oral Pathol Oral Radiol Endod. 1998; Scholar, the Scholar which may The clinical use and of in 2006; Scholar, and clinical exposure to bovine 2001; Scholar, exposure to bovine Med. Scholar, S. of to bovine A Med. 2001; 46: Scholar, B. C. Risk and clinical of by Pathol Med. 1998; Scholar, V. as a of exposure to bovine Scholar, of to bovine and human in two exposure to bovine J Scholar, from 1993; Scholar, of and risk in patients of 1993; Scholar, of to and with in a exposed to bovine Scholar, J.G. et of to bovine J 2001; Scholar This is why Dr. Choukroun developed PRF, to avoid this In and the of than use PRF. an the tube for PRF, we have that than tubes have been used in in maxillofacial, and that PRF has been used in at To this no has been at the of the French