• PLATELET-RICH PLASMA

    Par prpmed dans Accueil le 21 Juillet 2021 à 16:18

    From basic science to clinical applications

     

    For two decades, platelet-rich plasma (PRP) has been used in surgery to treat sports injuries. PRP contains growth factors and biologically active proteins that help in the healing of damaged tendons, ligaments, muscles and bones. This article highlights the current scientific knowledge of OLP and describes its application in sports medicine.

    Research into the biology of bone, ligament and tendon regeneration has led to the development of various products designed to help stimulate biological factors and promote healing. The use of autologous and recombinant products has rapidly expanded the ability of orthopedics to manipulate growth factors and secretory proteins to improve bone and soft tissue healing. But they are not used in the clinic, because many of these products have not been tested using rigorous scientific standards.

    Platelet-rich plasma (PRP) is one such example. This autologous product was first used and researched in the 70s of the last century. The healing properties of PRP have been used in clinical practice to increase the concentration of autologous growth factors and secretory proteins, which could enhance the regeneration processes at the cellular level. It is hoped that PRP enhances the recruitment, proliferation, and differentiation of cells involved in tissue regeneration. In the literature, PRP products, also known as PRP, platelet-rich concentrate, platelet gel or growth factor-rich preparation, have been investigated in in vitro and in vivo experiments in the field of maxillofacial and general surgery. Moreover, In orthopedic work, the role of PRP in the healing process of muscles and tendons has been investigated and it is becoming increasingly known to the general public. In February 2009, an article was published in the New York Times about treating an injury with a famous football player's OTP. Although there are numerous scientific studies.

    The use of OLT and related research in sports medicine and orthopedic surgery is not standardized and is mostly sporadic based on small case series (LE 4). Before introducing the OTP technique into the practice of sports medicine, it is necessary to evaluate the literature that supports the safety and effectiveness of this technique.

     

    Scientific knowledge of platelet-rich plasma

     

    What is OTP?

    Platelets are small, non-nuclear blood elements that play a major role in the processes of hemostasis. Platelets contain a variety of proteins, cytokines, and other bioactive factors that stimulate and regulate major links in injury healing. The normal number of platelets in the blood is in the range of 150,000 - 300,000 per microliter of whole blood. Plasma is the liquid part of the blood that contains clotting factors, other proteins and ions. Platelet-rich plasma is plasma containing about 1,000,000 platelets per microliter of plasma. PRP Tubes 3-5 times more growth factors than whole blood.

    Bioactive factors of PRP

    Platelet-rich plasma has the potential to improve healing through various growth factors and cytokines secreted from platelet α-granules. Major cytokines found in platelets include transforming growth factor β (TGF-β), platelet growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), fibroblast growth factor (FGF), epidermal growth factor, growth factor vascular endothelium (VEGF) and endothelial cell growth factor. These cytokines play an important role in the processes of cell proliferation, chemotaxis, differentiation, and angiogenesis (Table 1).

     
    Table 1 - Growth factors found in platelet-rich plasma and their physiological effects
    Factor Target Functions
    PD-EGF Blood vessel cells, outer skin cells, fibroblasts, and many other cell types Cell growth, recruitment, differentiation, skin wound closure, cytokine secretion
    PDGF A + B Fibroblasts, smooth muscle cells, chondrocytes, osteoblasts, mesenchymal stem cells Strong cell growth, recruitment, blood vessel growth, granulation, secretion of growth factors, collagen matrix formation  and bones involving bone morphogenetic proteins (BMP)
    TGF-β1 Blood vessel tissue, outer skin cells, fibroblasts, monocytes, TGF class including BMP, osteoblasts - highest level of TGF-βr Blood vessels (±), collagen synthesis , growth inhibition, apoptosis , differentiation, activation
    IGF-I, II Bone, blood vessels, skin, and other tissues; fibroblasts Cell growth, differentiation, recruitment, collagen synthesis with the participation of PDGF
    VEGF, ECGF Blood vessel cells Cell growth, migration, growth of new blood vessels c, antiapoptosis
    bFGF Blood vessels, smooth muscle, skin, fibroblasts, and other cell types Cell growth, cell migration, blood vessel growth
    PD-EGF - epidermal platelet growth factor, PDGF - platelet growth factor, BMP - bone morphogenetic protein, TGF - transforming growth factor, IGF - insulin-like growth factor, VEGF - vascular endothelial growth factor, ECGF - endothelial cell growth factor, bFGF - basic fibroblast growth factor.

     

    Biologically active factors of platelets are also contained in their dense granules. They contain serotonin, histamine, dopamine, adenosine and calcium ions. These factors are not growth factors, but they also play a fundamental role in the healing process. There are 3 stages of healing: inflammation, proliferation and reconstruction. The stage of inflammation begins immediately after injury, as a result of which platelets are activated, aggregate and secrete growth factors, cytokines and hemostatic factors necessary in the early stages of coagulation processes. Histamine and serotonin released by platelets activate macrophages and increase vascular permeability, which opens access to the focus of inflammation. Polymorphonuclear leukocytes migrate to the area of ​​inflammation and soon after, the cells begin to grow, while fibroblasts help to form the base substance. Through the activation of adenosine receptors, the regulation of inflammation and damage healing occurs (Tables 2 and 3).

     

      
    Table 2 - Biologically active molecules contained in platelet α-granules
    Factor categories Specific molecules Biological action
    Growth factors TGF-β Stimulates Matrix Synthesis
    PDGF Cell adhesion to the surface of chemoattractants, cell proliferation
    IGF-I, II Cell proliferation, maturation, bone matrix synthesis
    FGF Angiogenesis, fibroblast proliferation
    VEGF Cell proliferation
    EGF Angiogenesis
    ECGF Endothelial cell proliferation, angiogenesis
    Adhesive proteins Fibrinogen Blood clotting cascade (fibrin clot formation)
    Fibronectin Binding to integrins on the cell surface, effects on cell adhesion, cell growth, migration and differentiation
    Vitronectin Cell adhesion, chemotaxis
    Thrombospondin-1 Inhibition of angiogenesis
    Clotting factors Factor V, factor XI, protein S, antithrombin Everyone takes part in the activation of thrombin and, as a result, in the formation of a fibrin clot
    Fibrinolytic factors Plasminogen Plasmin precursor that breaks down fibrin
    Urokinase inhibitor Regulation of plasmin production
    α-2 antiplasmin Plasmin inactivation
    Proteases and antiproteases TIMP-4 Regulation of matrix degradation
    Metalloproteinase-4 Decomposition of the matrix
    α-1 antitrypsin Inhibition of a wide range of enzymes and proteinases
    Basic proteins Platelet factor 4 Inhibition of angiogenesis
    β-thromboglobulin Platelet activation, inhibition of angiogenesis
    Endostatin Inhibitors of endothelial cell migration and angiogenesis
    Membrane glycoproteins CD40 ligand Inflammation, synthesis of interleukins and integrins, adhesion of platelets to the endothelium, cellular signaling, modulation of interleukin-activated molecule-1 (PECAM-1) on leukocytes
    P-selectin Vascular endothelial adhesion molecule, assists in the binding and recruitment of leukocytes in the area of ​​inflammation
    TGF - transforming growth factor, PDGF - platelet growth factor, IGF - insulin-like growth factor, FGF - fibroblast growth factor, EGF - epidermal growth factor, VEGF - vascular endothelial growth factor, ECGF - endothelial cell growth factor, TIMP-4 - tissue inhibitor metalloproteinase-4.

     

    Table 3 - Biologically active molecules contained in dense granules of platelets
    Molecules Biological action
    Serotonin Vasoconstriction, increases capillary permeability, attraction of macrophages
    Histamine Increases capillary permeability, attraction and activation of macrophages
    Dopamine Regulation of heart rate and blood pressure, neurotransmitter
    ADP Induces platelet aggregation
    ATF Takes part in the reaction of platelets when they interact with collagen
    Ca 2+ Cofactor for platelet aggregation and fibrin formation 
    Catecholamines Sympathomimetic hormones are secreted by the adrenal glands in response to stress
    ADP - adenosine diphosphate, ATP - adenosine triphosphate.

    Platelets in PRP participate in thrombus formation, which contains a variety of cell adhesion molecules, including fibronectin, fibrin, and vitronectin. These molecules play an important role in the processes of cell migration and are of interest in the study of the bioactive properties of PRP. The blood clot itself may also play a role in the healing of the damage.

     

    Obtaining OTP

    Platelet-rich plasma can only be prepared from non-clotted blood. It cannot be prepared from clotted whole blood, from which serum is usually obtained, because most of the platelets remain in the resulting clot. Also, PRP cannot be prepared from serum, which is a clear liquid obtained from whole clotted blood and freed from cells and proteins involved in the clotting process. Serum contains very few platelets. For the preparation of PRP, blood is taken into a container with sodium citrate, which binds calcium ions, thereby blocking the entire coagulation cascade. This is followed by the centrifugation stage, which is carried out in one or two stages. In the first centrifugation, plasma and platelets are separated from red blood cells and white blood cells. Erythrocytes (7 microns in diameter) and leukocytes (7-15 microns in diameter) are much larger and heavier than platelets (2 microns in diameter) and are easily separated. The second centrifugation is carried out more gently and, as a result, OTP is concentrated; platelet-poor plasma remains in the supernatant.

    The next important step is the activation and aggregation of platelets, leading to the release of all biologically active factors contained in platelets. Several commercially available PRP systems use bovine thrombin as a clotting agent. In 10 minutes, about 70% of the bioactive factors contained in them are released from platelets, and in 1 hour almost 100%. However, the use of bovine thrombin can lead to complications associated with the formation of antibodies against it. This complication is very unlikely, but it is potentially possible and can lead to such a serious disease as immune-mediated coagulopathy. Also clots formed by thrombin show significant retraction.

    An alternative way to activate platelets is the use of a "fibrin matrix". The fibrin matrix is ​​formed from autologous fibrin , which is formed from fibrinogen under the action of its own thrombin, which is formed as a result of the addition of calcium chloride (CaCl2) to PRP. Calcium chloride is added before the second centrifugation, resulting in a dense fibrin matrix. Intact platelets interact with the formed fibrin network and are activated. This PRP activation technique is characterized by a low level of thrombin generated and, thus, minimization of platelet activation. As a result, platelets release growth factors rather slowly, and this process can take up to 7 days.

    The third way to activate PRP is the use of type II collagen . Collagen has been shown to be as effective as thrombin in stimulating platelet release of PDGF and VEGF growth factors. On the other hand, collagen- assisted clots are less retracted than thrombin- assisted clots .

     

    Effect of PRP on tissues

    Connective tissues such as tendons, ligaments, and muscles heal in three phases: inflammation, proliferation, and reconstruction. Various cytokines are actively involved in all of these phases. Cytokines play a major role in damage healing by interacting with transmembrane receptors on local and circulating cells, initiating intracellular signaling, which ultimately affects gene expression in the nucleus. As a result of this expression, proteins appear that regulate cell proliferation, cell chemotaxis, angiogenesis, cell differentiation, and extracellular matrix formation. It is known that cytokines and other biologically active factors isolated from PRP affect the main metabolic processes in the soft tissues of the musculoskeletal system, including tendons, ligaments and muscles.

    The effect of PRP on tendons. When evaluating the role of OLT in tendon repair, it is important to distinguish between acute injury and chronic tendinitis. ... Several recent studies have clearly shown that PRP positively influences gene expression and matrix synthesis in tendons and their cells. Incubation of cultured human tenocytes with PRP increases their proliferation and the total synthesis of collagen increases, slightly increases the expression of such enzymes of degradation of the extracellular matrix as metalloproteinase-1 and 3. the strength and rigidity of tendon callus (callus).

    The effect of PRP on muscles. Several cytokines contained in PRP have a positive effect on the healing of damaged muscles. For example, in a model of rupture of the gastrocnemius muscle in mice, basic fibroblast growth factor (bFGF) and IGF-I improve muscle healing. Autologous serum administered 2, 24, or 48 hours after injury to the gastrocnemius muscle in mice accelerated concomitant cellular activation and increased the diameter of the myofibril regeneration zone.

     

    Clinical use of platelet-rich plasma

     

    The literature has accumulated a large amount of data on the clinical use of OLT in such areas as maxillofacial surgery, otolaryngology, plastic surgery and general surgery.

     

    This article is a partial translation of an article by Timothy E. Foster, Brian L. Puskas, Bert R. Mandelbaum, Michael B. Gerhardt, and Scott A. Rodeo - Platelet-Rich Plasma: From Basic Science to Clinical Applications from The American Journal of Sports Medicine - November 2009 37: 2249-2251. From the original article, you can learn about the clinical use of PRP for injuries such as lateral epicondylitis and other tendinopathies of the elbow , tendinopathies of the Achilles tendon, patella , treatment of fractures, osteoarthritis , acute injuries of ligaments and muscles ; use of PRP during operations for reconstruction of the anterior cruciate ligament , restoration of the Achilles tendon, rotator cuff of the shoulder, articular cartilage . And you will also read detailed practical recommendations on the preparation method and the procedure for introducing PRP.