Francesco Inchingolo

Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF).

Platelet concentrates for surgical use are tools of regenerative medicine designed for the local release of platelet growth factors into a surgical or wounded site, in order to stimulate tissue healing or regeneration. Leukocyte content and fibrin architecture are 2 key characteristics of all platelet concentrates and allow to classify these technologies in 4 families, but very little is known about the impact of these 2 parameters on the intrinsic biology of these products. In this demonstration, we highlight some outstanding differences in the growth factor and matrix protein release between 2 families of platelet concentrate: Pure Platelet-Rich Plasma (P-PRP, here the Anituas PRGF - Preparation Rich in Growth Factors - technique) and Leukocyte- and Platelet-Rich Fibrin (L-PRF, here the Choukrouns method). These 2 families are the extreme opposites in terms of fibrin architecture and leukocyte content. The slow release of 3 key growth factors (Transforming Growth Factor β1 (TGFβ1), Platelet-Derived Growth Factor AB (PDGF-AB) and Vascular Endothelial Growth Factor (VEGF)) and matrix proteins (fibronectin, vitronectin and thrombospondin-1) from the L-PRF and P-PRP gel membranes in culture medium is described and discussed. During 7 days, the L-PRF membranes slowly release significantly larger amounts of all these molecules than the P-PRP gel membranes, and the 2 products display different release patterns. In both platelet concentrates, vitronectin is the sole molecule to be released almost completely after only 4 hours, suggesting that this molecule is not trapped in the fibrin matrix and not produced by the leukocytes. Moreover the P-PRP gel membranes completely dissolve in the culture medium after less than 5 days only, while the L-PRF membranes are still intact after 7 days. This simple demonstration shows that the polymerization and final architecture of the fibrin matrix considerably influence the strength and the growth factor trapping/release potential of the membrane. It also suggests that the leukocyte populations have a strong influence on the release of some growth factors, particularly TGFβ1. Finally, the various platelet concentrates present very different biological characteristics, and an accurate definition and characterization of the different families of product is a key issue for a better understanding and comparison of the reported clinical effects of these surgical adjuvants.

In Search of a Consensus Terminology in the Field of Platelet Concentrates for Surgical Use: Platelet-Rich Plasma (PRP), Platelet-Rich Fibrin (PRF), Fibrin Gel Polymerization and Leukocytes

In the field of platelet concentrates for surgical use, most products are termed Platelet-Rich Plasma (PRP). Unfortunately, this term is very general and incomplete, leading to many confusions in the scientific database. In this article, a panel of experts discusses this issue and proposes an accurate and simple terminology system for platelet concentrates for surgical use. Four main categories of products can be easily defined, depending on their leukocyte content and fibrin architecture: Pure Platelet-Rich Plasma (P-PRP), such as cell separator PRP, Vivostat PRF or Anituas PRGF; Leukocyteand Platelet-Rich Plasma (L-PRP), such as Curasan, Regen, Plateltex, SmartPReP, PCCS, Magellan, Angel or GPS PRP; Pure Plaletet-Rich Fibrin (P-PRF), such as Fibrinet; and Leukocyte- and Platelet-Rich Fibrin (L-PRF), such as Choukrouns PRF. P-PRP and L-PRP refer to the unactivated liquid form of these products, their activated versions being respectively named P-PRP gels and L-PRP gels. The purpose of this search for a terminology consensus is to plead for a more serious characterization of these products. Researchers have to be aware of the complex nature of these living biomaterials, in order to avoid misunderstandings and erroneous conclusions. Understanding the biomaterials or believing in the magic of growth factors ? From this choice depends the future of the field.

Current Knowledge and Perspectives for the Use of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF) in Oral and Maxillofacial Surgery Part 2: Bone Graft, Implant and Reconstructive Surgery

Platelet concentrates for surgical use are innovative tools of regenerative medicine, and were widely tested in oral and maxillofacial surgery. Unfortunately, the literature on the topic is contradictory and the published data are difficult to sort and interpret. In bone graft, implant and reconstructive surgery, the literature is particularly dense about the use of the various forms of Platelet-Rich Plasma (PRP) - Pure Platelet-Rich Plasma (P-PRP) or Leukocyte- and Platelet-Rich Plasma (L-PRP) - but still limited about Platelet-Rich Fibrin (PRF) subfamilies. In this second article, we describe and discuss the current published knowledge about the use of PRP and PRF during implant placement (particularly as surface treatment for the stimulation of osseointegration), the treatment of peri-implant bone defects (after peri-implantitis, during implantation in an insufficient bone volume or during immediate post-extraction or post-avulsion implantation), the sinuslift procedures and various complex implant-supported treatments. Other potential applications of the platelet concentrates are also highlighted in maxillofacial reconstructive surgery, for the treatment of patients using bisphosphonates, anticoagulants or with post-tumoral irradiated maxilla. Finally, we particularly insist on the perspectives in this field, through the description and illustration of the use of L-PRF (Leukocyte- and Platelet-Rich Fibrin) clots and membranes during the regeneration of peri-implant bone defects, during the sinus-lift procedure and during complex implant-supported rehabilitations. The use of L-PRF allowed to define a new therapeutic concept called the Natural Bone Regeneration (NBR) for the reconstruction of the alveolar ridges at the gingival and bone levels. As it is illustrated in this article, the NBR principles allow to push away some technical limits of global implant-supported rehabilitations, particularly when combined with other powerful biotechnological tools: metronidazole solution, adequate bone substitutes and improved implant designs and surfaces (for example here AstraTech Osseospeed or Intra-Lock Ossean implants). As a general conclusion, we are currently living a transition period in the use of PRP and PRF in oral and maxillofacial surgery. PRPs failed to prove strong strategic advantages that could justify their use in daily practice, and the use of most PRP techniques will probably be limited to some very specific applications where satisfactory results have been reached. Only a few simple, inexpensive and efficient techniques such as the L-PRF will continue to develop in oral and maxillofacial surgery in the next years. This natural evolution illustrates that clinical sciences need concrete and practical solutions, and not hypothetical benefits. The history of platelet concentrates in oral and maxillofacial surgery finally demonstrates also how the techniques evolve and sometimes promote the definition of new therapeutical concepts and clinical protocols in the todays era of regenerative medicine.

Current Knowledge and Perspectives for the Use of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF) in Oral and Maxillofacial Surgery Part 1: Periodontal and Dentoalveolar Surgery

Platelet concentrates for surgical use are innovative tools of regenerative medicine, and were widely tested in oral and maxillofacial surgery. Unfortunately, the literature on the topic is contradictory and the published data are difficult to sort and interpret. In periodontology and dentoalveolar surgery, the literature is particularly dense about the use of the various forms of Platelet-Rich Plasma (PRP) - Pure Platelet-Rich Plasma (P-PRP) or Leukocyte- and Platelet-Rich Plasma (L-PRP) - but still limited about Platelet-Rich Fibrin (PRF) subfamilies. In this first article, we describe and discuss the current published knowledge about the use of PRP and PRF during tooth avulsion or extraction, mucogingival surgery, Guided Tissue Regeneration (GTR) or bone filling of periodontal intrabony defects, and regeneration of alveolar ridges using Guided Bone Regeneration (GBR), in a comprehensive way and in order to avoid the traps of a confusing literature and to highlight the underlying universal mechanisms of these products. Finally, we particularly insist on the perspectives in this field, through the description and illustration of the systematic use of L-PRF (Leukocyte- and Platelet- Rich Fibrin) clots and membranes during tooth avulsion, cyst exeresis or the treatment of gingival recessions by root coverage. The use of L-PRF also allowed to define new therapeutic principles: NTR (Natural Tissue Regeneration) for the treatment of periodontal intrabony lesions and Natural Bone Regeneration (NBR) for the reconstruction of the alveolar ridges. In periodontology, this field of research will soon find his golden age by the development of user-friendly platelet concentrate procedures, and the definition of new efficient concepts and clinical protocols.

Shedding light in the controversial terminology for platelet-rich products: Platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet-leukocyte gel (PLG), preparation rich in growth factors (PRGF), classification and commercialism

A recent series of letters were published in JBMR-A about platelet concentrates for surgical use, where both terminology and content of these materials were hotly debated. The definition and classification of the platelet concentrate products are indeed very important issues, as many misunderstandings are widely spread in the large literature on this topic. These techniques were initially gathered under the name ‘‘platelet-rich plasma (PRP),’’ in reference to the generic term used in transfusion hematology, but this name is too general for the qualification of the many products developed now. In the first letter, Everts et al. insisted on the presence of leukocytes in most platelet preparations for surgical use. These authors explained a very important truth that many PRPs were in fact leukocyteand platelet-rich plasmas (LPRPs), and that the presence of leukocytes in these surgical adjuvants may be highly beneficial. They thus, introduced the term of ‘‘platelet-leukocyte–rich plasma (P-LRP).’’ Moreover, they pointed out that the two activation forms of the product (liquid platelet suspension or gelified fibrin-platelet clot) have different characteristics, and that the concentrates activated with a fibrinogen-cleaving agent (thrombin, batroxobin) should be named in fact as ‘‘platelet-leukocyte gels (PLG).’’ In this letter, these authors resumed the clarification process of the platelet concentrate definitions started in 2006. However, their proposals for terminology were not complete and have been improved and systematized in the recent publication of a wide classification system for these products. The first concern is that all PRPs do not contain leukocytes. Many PRPs obtained from cell separator units or from the Anitua’s preparation rich in growth factors (PRGF) subfamilies do not contain leukocytes and were classified as pure PRP (P-PRP). On the contrary, PRPs containing leukocytes were classified as L-PRP: this acronym seems obviously more logical and reader-friendly than P-LRP, but we agree that a consensus should be found to solve this issue once for good. The second issue is related to the gel form terminology. ‘‘Platelet gel’’ and ‘‘PLG’’ are too general terms. Indeed, products with a high-density fibrin network also exist and were classified as ‘‘platelet-rich fibrin (PRF),’’ some with leukocytes [leukocyteand platelet-rich fibrin (L-PRF)] and some without leukocytes [pure platelet-rich fibrin (P-PRF)]. All these PRFs are only available in the form of a very dense fibrin gel, while PRP gels are never so strong and dense. We thus believe that the activated form of P-PRP or L-PRP should simply be named ‘‘P-PRP gel’’ and ‘‘L-PRP gel’’ to differentiate them from the products of the PRF families. In the second letter, Anitua et al. agreed that the recent development of many different techniques with various platelet and leukocyte contents led to a confusing jungle of terms and products. This notion of ‘‘jungle of platelet concentrates’’ was already pointed out some years ago, when the main confusion between PRPs and the first PRF appeared. Anitua et al. were right in their call for the definition of a relevant terminology but their approach was unfortunately partisan. First, Anitua et al. claimed that leukocytes should be avoided in platelet concentrates for surgical use, to avoid the proinflammatory effects of the proteases and acid hydrolases contained in white blood cells, particularly when injected in tendons. However, these authors did not justify their statement with scientific evidence; to sustain their claim, Anitua et al. cited Ref. 9 describing very positive anabolic effects on tendon cells obtained with a PRP, . . . but the PRP described in this study was in fact a leukocyte-rich PRP. This question of the leukocyte content within platelet concentrates for surgical use is in fact an old debate. There is however actually no proof that the leukocytes within these surgical preparations might have undesirable side effects. On the contrary, several studies showed that L-PRPs have antimicrobial effects, but no undesirable inflammatory reactions have been observed with L-PRPs

Platelet Rich Fibrin (P.R.F.) in reconstructive surgery of atrophied maxillary bones: clinical and histological evaluations.

Introduction. Maxillary bone losses often require additional regenerative procedures: as a supplement to the procedures of tissue regeneration, a platelet concentrate called PRF (Platelet Rich Fibrin) was tested for the first time in France by Dr. Choukroun. Aim of the present study is to investigate, clinically and histologically, the potential use of PRF, associated with deproteinized bovine bone (Bio-Oss), as grafting materials in pre-implantology sinus grafting of severe maxillary atrophy, in comparison with a control group, in which only deproteinized bovine bone (Bio-Oss) was used as reconstructive material. Materials and Methods. 60 patients were recruited using the cluster-sampling method; inclusion criteria were maxillary atrophy with residual ridge < 5mm. The major atrophies in selected patients involved sinus-lift, with a second-look reopening for the implant insertion phase. The used grafting materials were: a) Bio-Oss and b) amorphous and membranous PRF together with Bio-Oss. We performed all operations by means of piezosurgery in order to reduce trauma and to optimize the design of the operculum on the cortical bone. The reopening of the surgical area was scheduled at 3 different times. Results. 72 sinus lifts were performed with subsequent implants insertions. We want to underline how the histological results proved that the samples collected after 106 days (Early protocol) with the adding of PRF were constituted by lamellar bone tissue with an interposed stroma that appeared relaxed and richly vascularized. Conclusions. The use of PRF and piezosurgery reduced the healing time, compared to the 150 days described in literature, favoring optimal bone regeneration. At 106 days, it is already possible to achieve good primary stability of endosseous implants, though lacking of functional loading.

Non-Hodgkin lymphoma affecting the tongue: unusual intra-oral location

IntroductionThe expression non Hodgkin lymphoma is used to cover a wide group of lymphoid neoplasias unrelated to Hodgkins disease, due to the huge histological variety and the tendency to affect organs and tissues that does not physiologically contain lymphoid cells.The intraoral location is not frequent (3 - 5 percent of cases) and the initial manifestations of the disease rarely take place here.Case presentationWe describe the case of a 73 years old Italian caucasian male who came to our attention with a tongue lesion. The clinical manifestation was macroglossia and bleeding, probably deriving from the tongue-bite injuries.The patient had been complaining of dyspnea for 48 hours.ConclusionA tongue affected by non-Hodgkins lymphoma rarely occurs. In spite of this, this possibility should always be considered for the differential diagnosis of benign and malignant lesions affecting such area.A rapid diagnostic assessment, together with an adequate histopathologic verification, are indeed essential to improve the management and the prognosis of this disease.

Influence of endodontic treatment on systemic oxidative stress.

Introduction: An increased production of oxidizing species related to reactive oral diseases, such as chronic apical periodontitis, could have systemic implications such as an increase in cardiovascular morbidity. Based on this consideration, we conducted a prospective study to assess whether subjects affected by chronic periodontitis presented with higher values of oxidative stress than reference values before endodontic treatment, and whether endodontic treatment can reduce the oxidative imbalance and bring it back to normal in these subjects. Materials and methods: The authors recruited 2 groups of patients from private studies and dental clinics: these patients were recruited randomly. The oxidative balance in both patients with chronic apical periodontitis (CAP) and healthy control patients was determined by measuring the oxidant status, using an identification of the reactive oxygen metabolites (d-ROMs) test, while the antioxidant status in these patients was determined using a biological antioxidant potential (BAP) test. Both these tests were carried on plasma samples taken from enrolled patients. Values were measured both before the endodontic treatment of the patients with chronic apical periodontitis, and 30 and 90 days after treatment, and compared to those obtained from healthy control patients. Results: It was found that, on recruitment, the patients with chronic apical periodontitis exhibited significantly higher levels of oxidative stress than control patients, as determined by the d-ROMs and BAP tests. Furthermore, the d-ROMs test values were shown to decrease and the BAP test values to increase over time in patients with chronic apical periodontitis following endodontic therapy. As the levels of oxidative stress in these patients tended to reduce and return to normal by 90 days following treatment. Conclusions: This study has demonstrated a positive association between chronic apical periodontitis and oxidative stress. Subjects affected by chronic apical periodontitis are exposed to a condition of oxidative stress, which is extremely dangerous to general health. Moreover, one can infer from these findings that through proper endodontic therapy, a good oxidative balance can be restored, thereby avoiding the risk of contracting the abovementioned diseases.

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 traps

To the Editor: We recently read with a great interest an article published in OOOOE, where He et al. 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, and the method and terminology used by He et al. raise several questions that require discussion. In their study, He et al. decided to incubate PRF and PRP in culture conditions for 28 days and to collect their exudates at 5 experimental times: 1, 7, 14, 21, and 28 days. The authors then tested the effects of these exudates on cell cultures. The advantage of this approach is to limit interspecies immune reactions between human PRP/PRF and rat cell cultures. The problem is that the authors tested the effects only of the growth factors and proteins in suspension in the PRP/ PRF exudate and not the true effects of the whole fibrin product. This is a significant limitation, because PRF is a dense fibrin-based solid biomaterial with a specific matrix architecture and cell content (platelet aggregates, activated leukocytes), and its exudate is clearly not the most interesting part of the product. Similarly, even if most PRPs are homogeneous liquid platelet suspensions, their exudates are not the sole bioactive components, because platelet aggregates themselves and their associated fibrin gel also trigger many biological mechanisms. Regarding the PRP, the authors claimed to use the Curasan method (Kleinostheim, Germany), but unfortunately they did not evaluate the leukocyte content of their final product. Curasan PRP is indeed classified in the leukocyteand platelet-rich plasma (L-PRP) family. Leukocytes are turntables of the growth factor release and play a significant role in the biology of these products. Moreover, the authors did not point out how and when they activated their PRP (using thrombin or batroxobin), and it seems that the PRP was in fact never activated into a gel. This approach is debatable, because the true final product used in patients is an activated L-PRP gel (in most cases), not an unactivated platelet suspension. Regarding the PRF, there are very serious questions about the protocol described by He et al. Indeed, the authors claimed to use the “PCCS protocol” (Platelet Concentrate Collection System; 3I, Palm Beach Gardens, FL, USA) to produce PRF, and referred to our own references about the PRF technology. The problem is that Choukroun’s PRF can not be produced with the PCCS device, because that centrifuge and tubes are specifically designed for the automated production of an L-PRP. Moreover, the authors claimed to have quantified the platelets in PRF, but that is impossible. Indeed, the production concept of PRF is based on the activation of platelets during the centrifugation to constitute strong fibrin/platelets strands and a dense final PRF clot; therefore, PRF exists only in a solid material form, never in a liquid form. An accurate and direct quantification of the platelet content is impossible, and we showed only recently and indirectly by soustraction that 95% of the platelets from the initial blood harvest were merged in the PRF clot. Consequently, we have no clear idea about the “PRF” the authors claimed to use in their study. PRF is a free and open-access technique. However, the name PRF is a trademark and refers to a specific preparation kit (PC02 centrifuge and collection kits; Process, Nice, France), production process, and final product. This trademark protection was not performed for industrial reasons, but to identify clearly the technique and protect it from confusion or scientific misunderstandings. Because there is no significant commercial interest in selling PRF devices, the only way to protect PRF was to force people to respect the definition of the product and thus to respect the trademark. In this article, it seems that the tested product was a liquid platelet concentrate produced with the PCCS, and maybe not an original Choukroun’s PRF. Care should be taken to respect a clear and reliable terminology to avoid significant misunderstandings in the literature. Finally, testing the in vitro effects of complex living biomaterial such as Choukroun’s PRF clot or membrane requires the greatest caution to get reliable results. Whatever the method, it is important to test the whole PRF and not just fragments, because PRF presents a specific 3-dimensional architecture and cell distribution. Moreover, care must be taken to always keep PRF in an osmotic medium (such as culture me-

Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in human cell cultures: Growth factor release and contradictory results

easier for PRF experiment. However, economical and ethical issues prohibit the use of large numbers of large animals for these experiments. In addition, the authors claimed that true Choukroun’s PRF could fill a large defect without the need for filling materials in humans. However, there are few references pertaining to Choukroun’s PRF only as a bone-filling material. Most references involved the use of Choukroun’s PRF for soft tissue augmentation. The use of fibrin-based material only as a filling material for large bone defects remains controversial. In summary, the failure to produce true Choukroun’s PRF from the rabbit is largely due to the low technical quality of the procedure. We can give 6 detailed technical tips as follows: 1) Select New Zealand white rabbits; 2) use sedation before sampling; 3) if possible, use a vessel dilatant; 4) consider an EDTA-pretreated conical tube to prevent immediate blood adherence to the tube wall; 5) use arterial blood if the sampling amount exceeds 5 mL; and 6) do not use a vacuum tube such as a glass tube or glass-coated tube that may be designed for humans, because it may collapse the rabbit’s fragile vessel and may also have a low success rate. The required amount of PRF in each experimental design must be tailored to the defect size; this will determine the required amount of blood sampling.