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Current Pharmaceutical Biotechnology, 2012, 13, 1121-1130 1121

1873-4316/12 $58.00+.00 © 2012 Bentham Science Publishers

Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF): Surgical Adjuvants, Preparations for In Situ Regenerative Medicine and Tools for Tissue Engineering

Tomasz Bielecki1,*

and David M. Dohan Ehrenfest2

1Department and Clinic of Orthopaedics, Medical University of Silesia, Sosnowiec, Poland;

2LoB5 unit, Chonnam Na-

tional University School of Dentistry, Gwangju, South Korea

Abstract: The recent developement of platelet concentrate for surgical use is an evolution of the fibrin glue technologies

used since many years. The initial concept of these autologous preparations was to concentrate platelets and their growth

factors in a plasma solution, and to activate it into a fibrin gel on a surgical site, in order to improve local healing. These

platelet suspensions were often called Platelet-Rich Plasma (PRP) like the platelet concentrate used in transfusion medi-

cine, but many different technologies have in fact been developed; some of them are even no more platelet suspensions,

but solid fibrin-based biomaterials called Platelet-Rich Fibrin (PRF). These various technologies were tested in many dif-

ferent clinical fields, particularly oral and maxillofacial surgery, Ear-Nose-Throat surgery, plastic surgery, orthopaedic

surgery, sports medicine, gynecologic and cardiovascular surgery and ophthalmology. This field of research unfortunately

suffers from the lack of a proper accurate terminology and the associated misunderstandings, and the literature on the

topic is quite contradictory. Indeed, the effects of these preparations cannot be limited to their growth factor content: these

products associate many actors of healing in synergy, such as leukocytes, fibrin matrix, and circulating progenitor cells,

and are in fact as complex as blood itself. If platelet concentrates were first used as surgical adjuvants for the stimulation

of healing (as fibrin glues enriched with growth factors), many applications for in situ regenerative medicine and tissue

engineering were developed and offer a great potential. However, the future of this field is first dependent on his coher-

ence and scientific clarity. The objectives of this article is to introduce the main definitions, problematics and perspectives

that are described in this special issue of Current Pharmaceutical Biotechnology about platelet concentrates.

Keywords: Blood platelet, fibrin, growth factors, leukocytes, platelet-rich fibrin (PRF), platelet-rich plasma (PRP), regenera-tive medicine, tissue engineering.

1. COAGULATION AS A PHARMACEUTICAL BIO-TECHNOLOGY: FROM FIBRIN GLUES TO PLATE-

LET CONCENTRATES

Improving healing is a constant issue in all surgical dis-ciplines, and the development of advanced biomaterials and pharmaceutical preparations has always influenced the his-tory of surgery. For example, improved implant surfaces considerably reduced the time of osseointegration [1] and the local use of antibiotics may protect and improve the healing of bone grafts [2]. Surgical adjuvants for topical use are an-other category of these biotechnologies designed for the lo-cal improvement of healing.

Wound healing is completely dependent on the initial mechanisms of hemostasis. When an organism is wounded, the first tissue to react is the circulating tissue: the blood. The wound triggers a cascade of reactions leading to the sealing of the vascular breach with platelets aggregates. But platelets not only stop the hemorragy by plugging the dam-aged tissue, these corpuscules also prepare the upcoming steps of tissue regeneration. Platelets deliver on the wounded sites a massive load of fibrinogen and enzymes, and also

*Address correspondence to this author at the Department and Clinics of

Orthopaedics, Medical University of Silesia, Sosnowiec, Poland; E-mails: [email protected]; [email protected]

release an enormous amounts of various molecules, particu-larly growth factors. While platelet growth factors stimulate cells of the wounded tissue in order to attract them on the site for tissue regeneration, platelet fibrinogen and circulat-ing fibrinogen start to polymerize into a dense fibrin network in order to glue and close the wound with a solid wall. The fibrin matrix is the final purpose of this complex cascade of reactions: the coagulation [3].

However, the coagulation should not be considered as a simple reinforcement of the anti-hemorrhagic platelet clot. Coagulation leads to the quick assembly of a brand new solid tissue, based on a dense fibrin matrix, cemented by platelets, and populated with leukocytes. From a philosophical stand-point, coagulation is the mechanism that allows the circulat-ing tissue to materialize into an adaptive and polymorphic solid form. From a practical standpoint, this quick-formed neo-tissue is the the first guiding matrix of healing, with the function of attracting more platelets and leukocytes, trapping circulating stem cells and allowing the migration and differ-entiation of the surrounding cells within the fibrin network. This matrix is then remodelled and transformed: this transi-tory tissue serves as the initial scheme for tissue regenera-tion.

In human sciences, we often try to mimic the efficient mechanisms observed in the Nature. The development of

1122 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 Bielecki and Ehrenfest

fibrin-based surgical adjuvants was therefore a logical and quite old approach. Since the first studies of Matras on the rat skin [4], these preparations have been tested in most clinical situations [5, 6], from plastic surgery (skin healing) to neurosurgery (as a guiding matrix for nervous regenera-tion) or orthopaedic surgery (to repair meniscal lesions, os-teochondral fractures or tendons for example). Results were more or less interesting, the purpose of these technologies was first to improve local angiogenesis and therefore to limit oedema, hematoma and pain. As the first matrix of healing, a strong fibrin bed could also promote early remodelling of the tissues and thus accelerate tissue regeneration on a wounded site [3, 7].

These glues were prepared from human blood in blood banks, and lyophilized products were marketed, with a very small - but not nil - risk of cross-contamination [6]. In many countries, the use of these fibrin products was therefore le-gally restricted. For this reason, many authors continued to test various methods for the preparation of autologous fibrin glues from the patient blood, but the technology required complex and time-consuming handlings and the help of a blood bank, several days before the surgical intervention where the product was supposed to be used: when Ta-yapongsak et al. described their autologous fibrin adhesive for maxillofacial bone grafting in 1994 [8], blood was har-vested 1 to 3 weeks before the intervention and required 2 days of handling before use. Moreover the biological and biomechnical properties of these fibrin products were lim-ited, because of their intrinsic nature: they were produced using the fibrinogen circulating in the blood plasma, polym-erization into fibrin being activated with bovine thrombin. However, in physiological situation, the true strength of a fibrin matrix and its properties are related to the interactions between circulating fibrinogen, platelet fibrinogen, the plate-let aggregates themselves and the many molecules released by the platelets [9]. Without the platelets, the fibrin glues were an incomplete imitation of the natural mechanisms, an incomplete imitation of the blood in its solid tissue form.

The logical evolution of the fibrin glues was therefore to incorporate platelets within the products [10-14]. For obvi-ous reasons of immune identity, such platelet enriched prod-ucts had to be autologous and produced with the patient’s own blood. The technology for the production of platelet concentrates already existed in blood banks and transfusion laboratories, and was quite similar to the technology for the preparation of autologous fibrin glues [10]: all these labora-tory techniques were unfortunately complex and time-consuming. However, contrarily to a platelet concentrate for transfusion purpose, the autologous platelet concentrates for topical use did not require to be perfectly purified: it just had to be safe and user-friendly. For this reason, many simplified techniques were developed, that could be handle easily in the surgical room, for the preparation of the product directly during the surgery.

2. PLATELET CONCENTRATE FOR SURGICAL USE:

PRINCIPLES AND TERMINOLOGY

In the first articles about platelet concentrates, various nomenclatures were proposed, but the term PRP (Platelet-Rich Plasma) became the reference after the founding article

of Marx et al. in 1998 [15]. PRP was simply the name of the platelet concentrates as defined in transfusion medicine, and this term was too general for the description of the many different products that could be used as platelet concentrates for surgical use. Several authors tried to improve the termi-nology in this field [16-18], and a consensus has still to be accepted and applicated in the literature on the topic [19].

Even if many different protocols for the production of platelet concentrates for surgical use were developed and sometimes marketed, all these technologies are based on the same basic principles [18]. In most techniques, the patient’s own blood is collected with anticoagulant, and centrifuged in order to separate the various components of the blood by their weight: red blood cells (RBCs), leukocytes, platelets, plasma. In most PRP technologies, there are 2 steps: the first centrifugation allows to separate the heavy RBCs from the other components, the second phase (often another centrifu-gation step) allows to separate the platelets and leukocytes from the acellular plasma (also called Platelet-Poor Plasma, PPP). The platelet and leukocyte layer is a whitish area, of-ten called the “buffy coat” (like in transfusion medicine). In many protocols, the buffy coat is collected and placed into suspension in a small volume of acellular plasma: this liquid mixture constitutes the PRP ready for clinical use.

The accurate separation of the platelets and leukocytes is difficult and requires an heavy ultracentrifuge (such as a cell separator for plasmapheresis) [20]. For this reasons, with most marketed protocols for platelet concentrate production, a significant amount of leukocytes is also in suspension in the final product. There is however a simple way to avoid the leukocyte collection: in some protocols, the buffy coat is not collected, and only the layers above are constituting the final platelet concentrate. Unfortunately, the logical consequence of this approach is that a significant quantity of platelets are also discarded (like in the PRGF, Plasma Rich in Growth Factor) [21].

This suspension of platelets can be injected without acti-vation (the platelets are activated on the wounded site natu-rally), for example in the treatment of tendons [22]; but in most applications it is activated with calcium chloride and/or bovine thombin (similarly to fibrin glue activation) in order to form a platelet-rich gel on the surgical site [23]. Compared to blood plasma, a commercial fibrin adhesive contains a 30-fold higher concentration of fibrinogen and factor XII (for the fibrin cross-linkage). With a small table centrifuge usable in daily clinical practice, it is obviously difficult to reach similar concentrations of circulating fibrinogen and factor XII from the patient’s own blood. However, platelets contain a significant quantity of fibrinogen and coagulation factors, and the gelling of a PRP is finally very similar to the gelling of a commercial fibrin adhesive.

In the literature, there are many variations in these tech-niques, in the way of discarding the RBCs after separation, or in the way to collect the small platelet concentrate layer, with or without the leukocytes [18]. Some procedures are automated using a blood collection device with 2 chambers, some others are manual using pipetting for the collection of the various layers obtained after centrifugation [24-27]. Moreover, initially, many techniques were discarding most of the acellular plasma, but nowadays, many authors recom-

PRP/PRF: Surgical Adjuvants, Preparations for in situ Regenerative Medicine Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. 7 1123

mend to use it also: since it is rich in growth factors and fi-brinogen, it can therefore be used as an autologous fibrin glue [23]. From a technical standpoint, the studies on the topic are disclosing very different blood centrifugation forces (from 160 g to 3000 g) and times (from 3 to 20 min for the first centrifugation step), and the definition of these parame-ters frequently seems to be empirical, without true care about the viability of the cells within the blood sample. Many PRP technologies are obviously not very accurate, and look like in-house cooking procedures. The cross-examination of these technical data is an impasse.

For these reasons, all these products should be character-ized and evaluated by their true content and architecture, and not by the way they were produced. Indeed there are many ways to get very similar products, and even many ways to prepare for clinical use the very same product: a more or less dilution of a platelet concentrate in an acellular plasma does not imply a true biological difference and, in a physiological environment with an important vascularization, the amount of growth factors of the product is less important than its structural characteristics. Researchers should therefore focus on the main intrinsic differences between the various prod-ucts in order to assess their properties.

Two key parameters define the various sorts of platelet concentrates [18]. The first parameter is the leukocyte con-tent. Unfortunately, the literature about platelet concentrates is very inconsistent about this aspect [16, 17], and many con-tradictory results published may be related to this key but neglected actors of healing. The role of these cells is slowly becoming an important theme of discussion and research [28], even if many authors still forget this parameter.

The second parameter is the fibrin architecture of the products. The PRP gels present an architecture similar to the former fibrin glues [29], with a light fibrin network and mostly tetramolecular or bilateral junctions fibrinogen po-lymerization (due to the artifical and quick polymerization with high thrombin concentrations). But the development of a second generation platelet concentrate technology, called PRF (Platelet-rich Fibrin)[30-34], highlighted that some products present higher fibrin concentration, stronger polym-erization (natural connected trimolecular or equilateral junc-tions) and higher biomechanical properties [35]. These PRF techniques are also based on the centrifugation of whole blood for the separation of its constituents, but the final ob-jective is to produce an activated fibrin-based biomaterial, and not a liquid platelet suspension. As previously explained the fibrin matrix is the purpose of coagulation, and the archi-tecture of the clot is probably more important than the growth factors themselves [35, 36]. The key role of the fibrin architecture has now to be assessed carefully [9].

Four families of products can thus be described, based on their leukocyte content and fibrin architecture [18]. Pure Platelet-Rich Plasma (P-PRP) and Leukocyte- and Platelet-Rich Plasma (L-PRP) are platelet suspensions, without or with leukocytes respectively; they are activated into respec-tively a P-PRP gel and a L-PRP gel using various plate-let/fibrinogen activators (e.g. bovine or autologous thrombin, calcium chloride or batroxobin), and present a light fibrin architecture. Pure Platelet-Rich Fibrin (P-PRF) and Leuko-cyte- and Platelet-Rich Fibrin (L-PRF) are activated platelet-

rich fibrin biomaterials, without or with leukocytes respec-tively, presenting a strong fibrin architecture. The activation can be artificial (like Fibrinet PRF, a P-PRF) [27] or natural (like Choukroun’s PRF, a L-PRF) [30].

This classification is logical and simple. But in order to be fully validated, it is necessary to evaluate the biological differences between these various products, particularly to prove that the growth factor release of these products is much more dependent on the fibrin architecture and the leu-kocyte content than on its initial platelet concentration [37].

In this special issue of Current Pharmaceutical Biotech-nology, a panel of experts proposes a consensus article about this key terminology issue, and in the following articles we try to give some prospective answers concerning the key role of fibrin structure and leukocytes.

3. A LARGE CLUSTER OF HEALING AND IMMUNE

CELLS AND MOLECULES

Blood is not only a solution delivering oxygen and nu-triments to the various cells and organs. Blood is a full tissue with interacting cells and matrix, with the specificity that this tissue assembles and stabilizes itself in a solid form only on a wounded site, during the clotting. From a philosophical standpoint, platelet concentrates for surgical use should therefore be considered as an activated and materialized form of blood, a solid version of the circulating tissue. Even if these products are often artificial version of the natural blood clot, they are supposed to mimic the intrinsic function of blood coagulation: to create the guiding matrix of tissue regeneration [3]. Platelet concentrates should thus be consid-ered as tissues, and not as pharmaceutical adjuvants, and the evaluation of the characteristics of these living biomaterials is as complex as the investigation of coagulation and healing itself.

Like all tissues, platelet concentrates gather many com-ponents acting in synergy and taking part in the healing process.

Platelets play a central role in hemostasis and healing processes. Upon their activation, platelet -granules release over 30 cytokines, including Platelet-Derived Growth Factor AB and BB (PDGF-AB and PDGF-BB), Transforming Growth Factor 1 and 2 (TGF- 1 and TGF- 2), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor 1 (IGF-1), and Epidermal Growth Factor (EGF), as well as active substances such as serotonin, catecholamines, von Willebrand factor, proaccelerin, osteonectin, and antimi-crobial proteins [38]. Indeed, platelets also have many func-tions in the antimicrobial host defense systems. These in-clude navigation toward the inflammatory chemoattractant N-Met-Leu-Phe, expression of immunoglobulin-G Fc recep-tors and for C3a/C5a complement fragments, and the capac-ity to generate antimicrobial oxygen metabolites including superoxide, hydrogen peroxide, and hydroxyl free radicals. Moreover, platelets interact directly with microorganisms, contribute to the clearance of pathogens from the blood-stream, and actively participate in antibody-dependent cell cytotoxicity against microbial pathogens [39-42]. Finally, platelets also contain dense granules, releasing large amounts of serotonin (5-HT), that may explain the pain reduction of-


ten encountered after clinical injection of platelet leukocyte gels [43].

The many platelet and leukocyte growth factors also pre-sent very specific biodynamics:

• PDGF is released by platelet granules but also macro-phages. This growth factor is a generalist healing booster, which stimulates cell proliferation, particularly fibroblasts and endothelial cells, in order to support tis-sue cell repopulation and neovascularization. It also in-duces the macrophage activation, required for wound cleaning and transforming these cells into a secondary source of growth factors [44, 45].

• TGF- 1 and - 2 are generalist stimulators of the con-nective tissue synthesis and remodelling, and are there-fore mediators of the cell differentiation. However, they also play a significant role in the chemotaxis and mito-genesis of connective tissue cells, such as fibroblasts and preosteoblasts, and also contribute to the inhibition of catabolic mechanisms (such as osteoclastic activity in bone resorption). TGF- are therefore key mediators of the tissue regeneration [46-48]. Platelet is an important source of TGF- 1, but these factors are synthetized by many different cell types, particularly the leukocytes [49].

• IGF-1 is both massively released by platelets and pre-sent in a circulating form in the blood stream. This fac-tor is therefore not only important during wound healing and clotting, but is a current mediator of the organism survival. Its function is highly dependent on the local situation, with a positive impact on cell proliferation and differentiation, but it is mainly the induction of survival signals protecting cells from many apoptotic stimuli [50-52].

Finally, one should remember that these growth factors never work alone, and that the final effect on cells is the re-sult of the final message given to the cells through their syn-ergic actions [51]. Moreover, all these growth factors can use autocrine, paracrine or juxtacrine pathways, and induce posi-tive feedback for their own production, leading to a sym-phony of reactions regulating the healing processes.

Many platelet concentrates also contain a substantial amount of leukocytes. Granulocytes and monocytes/ macro-phages have mainly a direct antimicrobial action, killing bacteria and destroying infectious molecules particularly by the use of oxidative derivatives produced by the myeloper-oxidase [16, 43]. They clean the wounded site from bacteria, dead cells and debris, but their growth factor releases also stimulate wound healing, chemotaxis for the recruitment of new leukocytes and neoangiogenesis (since new blood ves-sels are required to back up the host defensive cells)[53, 54]. However the lymphocytes may be in fact the most important leukocytes collected in a platelet concentrate, because these cells are not specialized in the massive and blind killing of bacteria, but are true regulating turntables of both immune reactions and healing, through their massive production of growth factors and signaling mediators [55].

The regulation of the VEGF production is a good exam-ple of the key role of the leukocytes in the growth factor bal-ance, from a quantitative and qualitative standpoint. Indeed,

platelets contain both angiogenesis stimulators, such as VEGF and basic Fibroblast Growth Factor (bFGF), and in-hibitors, such as endostatin and thrombospondin-1, in similar quantities with a local and limited regulation process (the different factors are often stored and released separately in order to avoid contradictory factor mediation)[56]. On the contrary, leukocytes produce considerable amounts of VEGF, and release it in different places and times following a global regulation mechanism.

Many other primordial components of the platelet con-centrates have yet to be analyzed, and this is one of the major issue in the near future. For example, apart from converting fibrinogen into fibrin, thrombin itself stimulates fibroblast proliferation, synthesis of various types of collagen and in-flammation mediators such as Interleukin-6 (Il-6). Consider-ing that PRPs are often activated with high concentrations of bovine thrombin, the effect of this component on the global product biology may be a key issue [57].

Another key actor of these products should also retain our attention. We recently localized progenitor stem cells in platelet concentrates; most of them were from the hema-topoietic lineage (CD34+/CD45+) and therefore may differ-entiate into mature endothelial cells and promote neoangio-genesis. But some of these cells were also from non-hematopoietic lineages (CD34+/CD45-) and could differen-tiate into mesenchymal cells (such as osteoblasts, chondro-cytes, etc). These progenitors cells probably play a signifi-cant role in the wound healing site. Moreover, the interac-tions of these circulating blood stem cells with the other ac-tivated components of the platelet concentrate are still com-pletely unknown [58]. This is an important field of research with potential biotechnological applications.

4. PRP AND PRF AS SURGICAL ADJUVANTS: A

WIDE CONCEPT WITH MANY APPLICATIONS

The careful examination of the many different actors assembled in a platelet concentrate allows to expect that these products will offer better healing properties than the fibrin glues still used in many surgical applications. And indeed, like fibrin glues many years ago, these technologies were recently tested in many clinical applications, such as oral and maxillofacial surgery [10, 15], Ear-Nose-Throat surgery [59], plastic surgery [23, 60, 61], orthopaedics and trauma surgery [43, 62-64], sports medicine [22, 65], general surgery [66, 67], gynecologic [68] and cardiovascular sur-gery [69] and even ophtalmology [70]. Historically, these technologies were first widely distributed in oral and maxil-lofacial surgery [10, 15], and the dental literature is very wide on this topic.

The platelet concentrates were mainly tested as surgical adjuvants, like the fibrin glues, with the objective to acceler-ate bone and soft tissue healing.

In bone surgery, the classical approach is to mix a bone graft with the platelet gel, and this is one of the first tested applications in maxillofacial surgery [15]. The fibrin matrix is expected to serve as a biological binder between the vari-ous bone blocks and to improve the development of the vas-cularization within the graft, while the growth factors are supposed to accelerate cell proliferation and migration (par-


ticularly endothelial cells for angiogenesis)[71]. Used as a surgical adjuvant, the platelet concentrates are in fact taking the function of an improved blood clot: indeed in bone sur-gery, bleeding of the surgical site is always expected, be-cause blood regenerative properties are strongly required for the good integration of a bone graft without necrosis se-questrum (this is an old but validated clinical principle). The use of platelet concentrate is somehow a way to mimic and amplify a natural phenomenon: blood coagulation for tissue regeneration.

In soft tissue surgery, the classical approach is to cover the surgical site with a wide layer of fibrin gel [23, 59, 72]. The platelet gel serves as a biological binder at the interface between the skin and the deep tissues (like a fibrin glue), and is also supposed to accelerate soft tissue healing, through angiogenesis stimulation and proliferation of the skin con-nective tissues. The main function of the platelet concentrate is therefore to protect the surgical site by stimulating the wound closure and avoiding local necrosis of the skin.

As surgical adjuvants, all the platelet concentrates follow similar concepts of clinical use: mixed with a bone graft or used as protective glue layer for soft tissues. The general philosophy of these preparations is to stimulate healing and reduce the risk of failure (particularly the necrosis of a bone graft or a cutaneous flap), but these products are sometimes also expected to improve the intrinsic quality of the treated tissues: stronger bone graft remodelling [34, 71] and gingival tissue maturation [36], invisible cutaneous scar [23], etc. This concept of improving tissue remodelling logically led to many other potential applications, such as mixing the platelet concentrate with fat grafts in order to stablize the grafted adipocytes [61, 73] or using the platelet gel as a protective cover for the bone graft [74, 75]. From a conceptual stand-point, these surgical adjuvants may be useful in all the sites where biological binder and a stimulation of angiogenesis are required. It is interesting to see that for this kind of appli-cations, fibrin and growth factors are both as important, and this could explain why the results are sometimes similar to the data published with fibrin glues.

Finally, the antimicrobial properties of these preparations are also very important characteristics that offer many collat-eral applications [76-78], such as local disinfection or con-tamination control of wounds [79]. These products could also act as regulators of the immune reactions, directly with their leukocyte content and growth factors [80], but also in-directly through the angiogenic properties of the fibrin ma-trix (early vascularization helps to drain oedema and in-flammation)[7].

The true effect of these products used a surgical adjuvant is still a source of debate. The published data are quite con-tradictory, some showing an improvement of bone and soft tissue healing, some proving the contrary. In all fields in fact, the literature is often difficult to sort and interpret because the tested products are simply not characterized, the leuko-cyte content and fibrin architecture being not described. Moreover, like in all trends, some teams were « for growth factors », while some others took a stand « against growth factors », and this partisan considerations obviously influ-ence the way data were acquired and published. This is a quite interesting irony of the History, since « growth fac-

tors » are often not the main constituents of these living bio-materials. Obviously, the properties of the various families of platelet concentrates are much more complex than the effects of a simple growth factor solution or the impact of a mono-constituent fibrin glue.

What could truly be expected from these products remain a hotly debated question. When using these products, there are so many parameters to consider that all statements should remain very careful. For example, when the application of a L-PRP with a bone allogeneic substitute induces mixed or bad clinical results, are these results induced by the intrinsic nature of this specific platelet concentrate family (such as an excessive leukocyte concentration and/or the overrepresenta-tion of macrophages ?), by the way the L-PRP was used (which method of activation ? Into or onto the graft ?), or by the interactions between a specific bone allograft with a spe-cific platelet concentrate (some osteochondral allografts pre-sent a natural strong immunogenic potential, that can trigger the overreaction of some overrepresented L-PRP leuko-cytes)[81-83] ? Most of the underlying mechanisms explain-ing the published clinical results are far from being eluci-dated: unfortunately, the right questions or hypotheses are often not even set out.

Finally, the validation of the various applications is a very long way, where a clarification of the tested products is a first very important step. However, platelet concentrates are not only surgical adjuvants. They can be a full treatment by themselves.

5. PRP AND PRF FOR IN SITU REGENERATIVE

MEDICINE

The concept of in situ regenerative medicine is to inject cells or pharmaceutical preparations with the objective to induce locally the regeneration of a tissue. It is a pharmaceu-tical concept, where platelet concentrates are no more a sur-gical adjuvant to the treatment: they become the treatment. This kind of application is an important trend in the field of platelet concentrates, because these preparations contain high concentrations of autologous cells and proteins (particularly growth factors) that could promote a local cell stimulation.

The first application based on this concept is to inject unactivated liquid platelet suspensions in various tissues in order to stimulate locally the cells and tissue regeneration. This non-surgical approach is particularly relevant with ten-dons or aged skin [22, 65], but also limited to minimally damaged tissues that do not require massive surgical repara-tion. In these applications, growth factors, leukocytes and platelet themselves are all expected to stimulate the cells, but it is difficult to determine which element of the « regenerative cocktail » is the most important. The clinical results have thus to be strongly correlated with an accurate and extensive characterization of the product (and not only the evaluation of the platelet concentration).

The second application is to use these products as solid biomaterials sustaining the release of regenerative molecules on a wounded site. PRP gels and PRF technologies allow to produce a significant volume of this fibrin-based biomaterial rich in many healing factors, particularly platelet growth factors. These solid volumes can not easily be injected in a


tissue, and should therefore be used like a bioactive bandage. The main limit to this approach is that these gels are soft and quite fragile, and can only be used in a very protected area with minimal biomechanical constraints: the integrity of the fibrin matrix, its cell content and its local release of growth factors must be protected [37, 84]. The best examples are the use of a L-PRF bandage on chronic skin ulcers or the use of these fibrin gels to fill a maxillary sinus cavity during dental implant placement in order to promote intra-sinus bone re-generation [85, 86]. In both cases, the fibrin matrix releases growth factors and stimulating molecules during a long pe-riod, while the leukocytes and platelets themselves contrib-ute to the local stimulation of healing [37, 84, 87, 88]. Moreover, the fibrin itself can serve as a temporary guide for tissue regeneration.

In the international literature, the use of PRP/PRF for in situ regenerative medicine is less controversial than their use as surgical adjuvants. The studies using this approach often highlight good results. This observation could be explained easily: these products were often tested as surgical adjuvants in clinical procedures that already showed good results, and where the product was only supposed to improve the local healing, to amplify the role of the natural bleeding. For in situ regenerative medicine, the platelet concentrates are used alone in situations where other treatments are often not very efficient. The positive effects are therefore easier to assess and not debatable.

6. PRP AND PRF AS TOOLS FOR TISSUE ENGI-

NEERING: A VISION OF THE FUTURE.

Regenerative Medicine is a quite general concept that also includes the process of creating functional tissues to repair or replace damaged tissue or organ function: this is tissue engineering, the Art of bringing together cells and supporting scaffolds in order to build a living tissue. Logi-cally, if platelet concentrates offer new opportunities for in situ regenerative medicine, these preparations raise a signifi-cant interest as tools for tissue engineering applications.

A first approach was to use a platelet lysate or the plate-let-poor plasma as a safe substitute for animal serum in cell-based therapy applications, particularly for the culture of mesenchymal stem cells [89-91]. Such a solution rich in growth factors can easily be produced in blood banks, start-ing from platelet concentrates used in transfusion medicine, and presents a limited immune imprint that allows to use platelet releasates and cells from different donors in culture. However the inconvenient is that this approach does not truly allow to use the full characteristics and potential of a platelet concentrate; it is only a substitution culture medium.

Another approach would be to use the full potential of the platelet concentrate as a stimulating culture medium, a biological binder and a supporting scaffold for cells. The concept is to build in vitro an engineered tissue using mesen-chymal stems cells growing in a platelet gel: the fibrin scaf-fold serves then as the physical support of the graft, without any additionnal solid material [92, 93]. The use of fibrin scaffolds for the implantation of stem cells is an old trend in tissue engineering [94-97], and the PRP gels offer an inter-esting autologous and growth factor-boosted alternative to the artificial fibrin matrix commonly tested in these applica-

tions [98]. Another version of this concept is to seed mesen-chymal stems cells on a first scaffold (for example an allo-geneic, xenogeneic or synthetic bone substitute) and to add a platelet gel as biological binder and growing medium [99-103]: this technique offers a stronger supporting scaffold and is relevant for the grafting of larger volumes.

These various options were mainly experimented for bone tissue engineering, and gave interesting preliminary clinical results [104-110]. However several authors also no-ticed than the best results were always obtained when the platelet gel was reinforced with a fibrin glue [111, 112]: in these tissue engineering applications, the platelets and growth factors alone are not sufficient, the key role of the fibrin matrix as a biological binder and supporting scaffold was once again confirmed.

The logical evolution of this approach is therefore to use products from the PRF subfamilies, in order to combine both the stimulating effects induced by the growth factors [37] and the support function of a dense fibrin matrix [35]. Recent studies also suggested that leukocytes may play a significant role in the in vitro cell development, since all cell cultures with a L-PRF are in fact cocultures with leukocytes [87, 88]. This observation reminds us of the strong immune identity of these products, and implies that primary cultures from the same patient as the platelet concentrate should be used when tissue engineering applications are considered with platelet concentrates in general, and leukocyte-rich products in par-ticular [113]. The logical consequence is that potential appli-cations of a P-PRP and a L-PRF, as tissue engineering tools for mesenchymal stem cells, are completely different: a P-PRP gel is an improved fibrin glue rich in growth factors, embedding the cells and supporting their immediate growth and implantation [92], while a L-PRF is a strong fibrin-based tissue that guides and regulates the proliferation/ differentia-tion balance of the culture and may constitute the regulation core of the engineered neo-tissue (particularly through the long term slow release of mediators and the interactions with the leukocytes) [37, 87, 88]. A P-PRP is a supporting pe-ripheric ingredient, a L-PRF is also a central organizing node.

Used as tissue engineering tools, the platelet concentrates present a great potential for the future. But these autologous products are much more difficult to handle than bank cell lineages or synthetic biomaterials, because they are intrinsi-cally polymorphic, adaptive and versatile. We have to adapt our technical procedures to their multiple characteristics, we can not accurately design them for a unique and specific function. For example, a L-PRF is an optimized blood clot: understanding the biomaterial is therefore as complex as understanding the circulating tissue and healing themselves. The intrinsic biology of these products should thus be care-fully assessed and defined, particularly their leukocyte con-tent and fibrin architecture, in order to define a solid charter of main principles to follow when using a platelet concen-trate in tissue engineering.

Finally, by extension, platelet concentrates could also be used as in vivo tissue engineering tools: it is for example the case when bone cells are collected in the patient and imme-diately mixed with a supporting scaffold, such as L-PRF


membranes and/or bone substitutes. Potential applications are numerous.

7. INTRODUCTIVE CONCLUSIONS.

Surgical adjuvants, preparations for in situ regenerative medicine and tools for tissue engineering: the platelet con-centrates can be used in all these applications. But the defini-tion of the best indications and methods for each product family remains a long-term project, where little is known and understood yet. The Art of reviewing published data is a very ingrate work when done seriously, and the world of the platelet concentrates is probably one of the most difficult to review. The lack of acceptable terminology, the frequent absence of basic characterization of the products and the many misunderstandings encountered in the literature [113-115] make meta-analysis of data almost impossible. Readers should be aware of these key issues when reading a review on the topic.

In this special issue of Current Pharmaceutical Biotech-nology, several experts try to highlight the beneficial impact of these therapies in different clinical fields. This issue starts with an important consensus conference chapter, where a global and simple terminology and classification of products are developed, and guide us all along this special issue. Piero Borzini and collaborators complete this classification by a discussion about the technical and economic aspects of the PRP technologies, from the standpoint of the blood transfu-sion specialist. Then, following the principles debated in the consensus conference article, we perform a first demonstra-tion of the significant differences between the various fami-lies of product in terms of biological kinetics, particularly growth factor release: the fibrin architecture and leukocyte content of the various products have a significant impact on their biology. Another following chapter finally describes the role of leukocytes from L-PRP/L-PRF in wound healing processes and immune defense.

The following chapters are mainly clinically oriented. Are all these technologies useful in gynecology, cardiac and general surgery ? Peter Everts and collaborators try to an-swer to this question, through their experience and the scarce literature on these clinical applications. Orthopaedics is also a field where the impact of the platelet concentrates was in-vestigated: Ting Yuan et al. discuss the relevance of the L-PRPs in trauma surgery and Allan Mishra and collaborators review the impact of these preparations in sport medicine. Sport medicine is indeed a field where the platelet concen-trates present excellent results of regenerative medicine, in clinical situations where other treatments failed to promote a complete recover. Matthias Zumstein et al. then particularly describe the key role of fibrin and leukocytes for the long-term delivery of growth factors in rotator cuff repair. How-ever, whatever the results reported in various fields of medi-cine, a very significant part of the PRP/PRF literature is re-lated to oral and maxillofacial surgery: Del Corso et al. re-view the current knowledge and perspectives of these prod-ucts in periodontal and dentoalveolar surgery, while Simon-pieri et al. particularly develop the applications in implantol-ogy, bone graft and maxillofacial reconstructive surgery. The 2 last chapters focus on specific applications with strong potential for in situ regenerative medicine: Alio et al. de-

scribe the use of PRPs in ophthalmological surgery, and Ci-eslik-Bielecka et al. discuss the current knowledge and per-spectives about the use of PRP and PRF in plastic surgery and for the management of skin chronic wounds. These latter applications for the treatment of skin ulcers are particularly efficient and present a great potential for the future.

This special issue is first of all a platform of debate and clarification for the future of the field. Indeed, all the pub-lished results have to be discussed carefully. The understand-ing of the basic mechanisms of these living biomaterials, and the awareness of the misunderstandings of the field, is more important than the detailed description of the - often incom-plete - published data. For these reasons, aside from review-ing the actual knowledge (or supposed knowledge), this spe-cial issue is also the occasion to describe wider principles and potential applications, sometimes well known but often not widely spread. Starting with a consensus article about terminology, the articles of this issue try to open doors for the future, point out the perspectives rising and draw the great lines for the development of these technologies and their clinical applications. Hopefully, with the many recent advances in terms of product characterization and terminol-ogy, we can hope that the data published in this field will be more accurate in the future.

DISCLOSURE OF INTEREST

The authors declare no competing financial interests.

ACKNOWLEDGEMENTS

This work was supported by the LoB5 Foundation for Research, Paris, France.

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Received: July 20, 2010 Accepted: September 06, 2010

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