Sean M. O'Connell, PhD, Richard J. Carroll, PhD, Andrew Beavis, MS, Steven P. Arnoczky, DVM.  Flow Cytometric Characterization of CASCADE Platelet-Rich Fibrin Matrix (PRFM): The Impact of Exogenous Thrombin on Platelet Concentrates (PC). Musculoskeletal Transplant Foundation, 2008.

Platelet-derived growth factors (GF) have been shown to be critically involved on the early (bFGF; PDGF; IGF) and later stages (EGF, VEGF, TGF-B, IGF) of the healing process in bone, cartilage and soft tissue repair. A variety of methods have been employed to harness autologous platelet growth factors as an aid to optimize healing in a number of therapeutic arenas, including orthopaedics. Most of these methods produce platelet-rich plasma (PRP) by first isolating and concentrating the platelets from whole blood. However, the platelet concentration (PC) is unstable and difficult to administer in a number of clinical applications. To produce a stable clot, calcium chloride together with an exogenous activator, such as thrombin, is used to activate the platelets and residual fibrin in the PC. Excess thrombin treatment is thought to result in irreversible platelet degranulation with loss of platelet integrity and immediate growth factor release.

 The CASCADE Autologous Platelet System was developed with the goal of preserving platelets and their associated GF to facilitate optimal tissue repair. The system achieves this goal by employing a novel strategy of concentrating both platelets and fibrin in dense cross-linked platelet-rich fibrin matrix (PRFM) without the use of excess exogenous activators such as thrombin. The lack of excess thrombin would tend to ensure platelet integrity during PRFM processing and allow a gradual release of GF's versus premature total release. Indeed, previous studies have demonstrated a gradual release of the platelet growth factors over a seven day time course indicating that the platelets remain functionally viable PRFM production in the Cascade system. In contrast, addition of exogenous thrombin (bovine, 1000U/mL) (BoThr) resulted in a rapid release of the total measured GF from the platelets with and without the addition of exogenous thrombin.

 Flow cytometry, together with fluorescence immuno-cytochemistry, was used to determine the extent of platelet activation, degranualtion and loss of physical integrity. Autologous platelets were isolated with the Cascade system, with and without the addition of exogenous BoThr. BoThr was chosen since it is the most common activator used to produce a PC. For flow cytometry, the platelets were suspended in solution and stained with fluorescently tagged monoclonal antibodies (MAbs) that recognize specific cell surface markers which distinguish between unactivated and activated platelets.

 These results, taken together with previous studies, reinforce the premise that the Cascade system, which does not require exogenous thrombin, provides enhanced tissue repair by isolation, concentration and preservation of autologous platelets in dense fibrin matrix (PRFM). This PRFM is able to deliver a sustained release of concentrated autologous growth factors to the repair site over the space of days and weeks, compared to thrombin activated PC preparations which release their growth factors within minutes and hours of application.

D.V. Brezniak, D.G. Moon, J.A Beaver and J.W Fenton II.  Haemoglobin inhibition of fibrin polymerization and clotting. Blood Coagulation and Fibrinolysis, Vol. 5, 1994. Pg. 139-143.

Clotting of human plasma by human a-thrombin was prolonged in the presence of haemoglobin as was human and bovine fibrinogen. Specifically, the clot time doubled for human plasma, human fibrinogen and bovine fibrinogen at 483, 233, and 116 uM haemoglobin, respectively. Fibrinopeptide A release was not inhibited at concentrations in ~16000 molar excess compared with a-thrombin. Turbidometric analysis of fibrin polymerization showed a lengthening of the lag phase as well as the fibrin assembly process in the presence of haemoglobin. These findings suggested that neither fibrinogen recognition nor catalytic efficiency of thrombin was affected, implying that haemoglobin interferes with fibrin polymerization. Since human blood contains sufficient haemoglobin in erythrocytes to generate concentrations of up to 2.3mM upon every cell lysis, and haemoglobin concentrations of 0.16-0.48 mM caused 1.25 to two times longer clotting times in fresh human plasma, respectively, haemoglobin may act to modulate clot formation under conditions of haemolysis.

 In the present study it was found that haemoglobin prolonged thrombin clotting of fibrinogen and was attributable to impaired fibrin polymerization.  Prolongation of the clotting time occurred when either human plasma or purified human fibrinogen (as well as bovine fibrinogen) were clotted in the presence of reduce haemoglobin.

Haemoglobin inhibited thrombin-induced clotting of both human plasma and purified human fibrinogen as well as bovine fibrinogen. The interference lay within the polymerization process as shown by the lengthening of the early phases of fibrin polymerization that are involved in formation of protofibrils as well as a decrease in the rate of lateral association.

These data indicate that one of the key components of the inflammatory ‘walling off' response (e.g., fibrin formation) can be significantly delayed at the local sites of tissue injury by the presence of lysed erythrocytes and free haemoglobin.

David M. Dohan Ehrenfest, Lars Rasmusson and Tomas Alberktsson Department of Biomaterials, Institute of Clinical Sciences, The Sahlgrenska Academy at University of Gothenburg, Sweden.  Classification of Platelet Concentrates: From Pure Platelet-rich Plasma (P-PRP) to Leucocyte-and Platelet-rich Fibrin(L-PRF). Trends in Biotechnology, Vol. 27 No. 3.

The topical use of platelet concentrates is recent and its efficiency remains controversial. Several techniques for platelet concentrates are available; however, their applications have been confusing because each method leads to a different product with different biology and potential uses. Here, we present classification of the different platelet concentrates into four categories, depending on the leucocyte and fibrin content: pure platelet-rich plasma(P-PRP), such as cell separator PRP, Vivostat  PRF ot Anitua's PRFG; leucocyte- and platelet-rich plasma (l-PRP), such as Curasan, Regan, Plateletex, SmartPReP, PCCS, Magellan or GPS PRP, pure platelet-rich fibrin(P-PRF), such as Fibrinet; and leucocyte- and platelet-rich fibrin (L-PRF), such as Choukroun's PRF.

 Leucocyte-poor and/or pure platelet-rich fibrin (P-PRF) concentrates

In this category, there is only one method available. The Fibrinet PRFM kit by Cascade Medical (New Jersey, USA)

The main difference is that only very low amounts of leucocytes are collected owing to the specific separator gel used in the method. However, the platelet collection efficiency is high and the preservation of the platelets during the procedure seems to be acceptable. Platelet activation and fibrin polymerization are triggered using only calcium chloride, the same method as in Anitua's PRGF protocol. However, the fibrin matrix in Fibrinet PRFM is denser and more stable than that in PRP's, probably due to the dynamic clotting during the second centrifugation step, which is more efficient than static PRP polymerization.

Visser, LC Caballero, O, Arnoczky, SP, Laboratory for Comparative Orthopaedic Research, Michigan State University, East Lansing, MI.  Platelet-Rich Fibrin Constructs Elute Higher Concentrations of TGF-B1 and Increase Tendon Cell Proliferation Over Time When Compared to Blood Clots: A Comparative In Vitro Analysis. Presentation: 2010 Orthopedic Research Society Meeting.

Platelet-rich plasma (PRP) is defined as an autologous concentration (above baseline) of platelets and their associated growth factors. Recently, use of the PRP has been proposed as a way to enhance and accelerate the repair of biologically compromised tissues. However, because of the short half-life of most growth factors, PRP may not be able to provide a prolonged release of chemotatic and mitogenic factors needed to initiate and sustain a healing response in biologically compromised tissue. A platelet-rich fibrin matrix (PRFMatrix) and platelet-rich fibrin membrane (PRFMem) may permit a sustained elution of growth factors through the entrapment of growth factor-laden platelets in a dense fibrin matrix. The purpose of this study was to compare the concentration of a sential growth factor (TGF-B1) eluted from PRFMatrix, PRFMem, and a whole blood clot (BC) over time.

A BC, PRFMatrix, and PRFMem of similar volumes were created from each of 4 adult beagle dogs. Each construct was placed in  individual tissue culture wells and submerged in serum-free media for 7 days. The conditioned media was collected and replenished with fresh media on days 1, 3, 5, and 7. The concentration of TGF-B1 (n=4/construct/time point) eluted into the media was measured with an ELISA.

At days 1 and 3, the eluent from both PRFMatrix and PRFMem contained significantly more TGF-B1 when compared to the BC eluent. Eluent from both PRFMatrix and PRFMem produced a significant increase in fibroblast proliferation compared to the BC at all time points examined (except the PRFMatrix at day 7). The PRFMem eluent caused a significant increase in cell proliferation over both BC and PRFMatrix eluent at all time points examined.

The results of this study show that the eluent from both the PRFMatrix and PRFMem produced an increase in growth factor concentration and tendon cell proliferation over time when compared to a blood clot of similar volume in vitro. Additional studies are needed to determine if the use of a PRFMatrix or PRFMem can enhance the repair of biologically compromised tissues in vitro.

E. Lucarelli, B. Dozza, M. Pierini, D. Donati. Dipartmento di Patologie Ortopediche-Traumatologiche, Rizzoli Orthopedic Institute, Bologna, Italy.  P.L. Tazzari, P.P. Pagliaro.  Orsola-Malpighi Hospital, Bologna, Italy.  S.M. O'Connell, R. Baretta.  Cascade Medical Enterprises, LLC.  F. Ricci, S. Sqarzoni, E.I Oprita.  A Recently Developed Bifacial Platelet-Rich Fibrin Matrix. European Cells and Materials, Vol. 20, 2010, pg. 13-23.

Platelet-rich plasma (PRP) is used clinically in liquid or gel form to promote tissue repair. Because of the poor mechanical properties, conventional PRP is often difficult to handle when used in clinical settings and requires secure implantation in a specific site, otherwise when released growth factors could be washed out during an operation. In this study, we analyzed the end product of a recently developed commercially available system (FIBRINET®) which is a dense pliable, platelet-rich fibrin matrix (PRFM). We characterized the mechanical properties of PRFM and tested whether PRFM releases growth factors and whether released growth factors induce the proliferation of mesenchymal stem cells (MSC). Mechanical properties as well as platelet distribution were evaluated in PRFM. PRFM demonstrated robust mechanical properties, with a tear elastic modulus of 937.3 ± 314.6kPa, stress at a break of 1476.0 ± 33.8%. PRFM maintained its mechanical properties throughout the testing process. Microscopic observations showed that the platelets were localized on one side of the matrix.

MSC culture media supplemented with 20% PRFM-CM stimulated MSC cell proliferation; at 24 hours and 48 hours the induction of the proliferation was significantly greater that the induction obtained with the media supplemented at 20% foetal bovine serum.

The present study shows that the production of a dense, physically robust PRFM made through high-speed centrifugation of intact platelets and fibrin in the absence of exogenous thrombin yields a potential tool for accelerating tissue repair.

Anitua E, Andia I, Ardanza B, Nurden AT.  Biotechnology Unit, IFR No 4, Hopital Cardiologique, Avenue Magellan, 33604 Pessac, France.  Autologous platelets as a source of proteins for healing tissue regeneration. Thromb. Haemost., 2004.

Platelets are known for their role in haemostasis where they help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate, and form a procoagulant surface leading to thrombrin generation and fibrin formation. Platelets also release substances that promote tissue repair and influence the reactivity of vascular and other blood cells in angiogenesis and inflammation. They contain storage pools of growth factors including PDGF, TGF-beta and VEGF as well as cytokines including proteins such as PF4 and CD40L. Chemokines and newly synthesized metabolites are also released. The fact that platelets secrete growth factors and active metabolites mean that their applied use can have a positive influence in clinical situations requiring rapid healing and tissue regeneration. Their administration in fibrin clot or fibrin glue provides an adhesive support that can confine secretion to a chosen site. Additionally, the presentation of growth factors attached to platelets and/or fibrin may result in enhanced activity over recombinant proteins. Dental implant surgery with guided bone regeneration is one situation where an autologous platelet-rich clot clearly accelerates ossification after tooth extraction and/or around titanium implants. The end result is both marked reductions in the time required for implant stabilization and the improved success rate. Orthopaedic surgery, muscle and/or tendon repair, reversal of skin ulcers, hole repair in eye surgery are other situations where autologous platelets accelerate healing. Our aim is to review these advances and discuss the ways in which platelets may provide such unexpected beneficial therapeutic effects.

Everts PA, Knape JT, Weibrich G, Shonberger JP, Hoffman J, Overdevest EP, Box HA, van Zundert A.  Department of Extra Corporeal Blood Management, Catharina Hospital, Eindhoven, The Netherlands. Platelet-rich Plasma and platelet gel: a review.  J Extra Corp Technol., June, 2006; 38(2):174-87.

Strategies to reduce blood loss and transfusion of allogeneic products during surgical procedures are important in modern times. The most important and well known autologous techniques are preoperative autologous predonation, hemodiliution, preoperative red cell salvage, postoperative wound blood autotransfusion, and pharmacologic modulation of the haemostatic process. At present, new developments in the preparation of preoperative autologous blood component therapy by whole blood platelet-rich plasma (PRP) and platelet-poor plasma (PPP) sequestration has evolved. This technique has been proven to reduce the number of allogeneic blood transfusions during open heart surgery and orthopedic operations. Moreover, platelet gel and fibrin sealant derived from PRP and PPP mixed with thrombin, respectively, can be exogenously applied to tissues to promote wound healing, bone growth, and tissue sealing. However, to our disappointment, not many well designed scientific studies are available, and many anecdotic stories exist, whereas questions remain to be answered. We therefore decided to study preoperative blood management in more detail with emphasis on the application and production of autologous platelet gel and the use of fibrin sealant. This review addresses a large variety of aspects relevant of platelets, platelet-rich plasma, and the application of platelet gel. In addition, an overview of recent animal and human studies is presented.