PRP Basic Science. Platelets. Definition of PRP 10/4/2011. Questions that this talk aims to answer

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1 PRP Basic Science Peter J. Moley, MD Hospital for Special Surgery October 5, 2011 Questions that this talk aims to answer 1. What is PRP? 2. What blood components are NOT in PRP? 3. What are the active ingredients in PRP? 4. How is PRP processed? Does it matter how it s processed? 5. What are the concentrations of platelets and growth factors in PRP preparations? Which concentrations are optimal? 6. Healing and mechanism of PRP Definition of PRP Platelet-rich plasma: Portion of the plasma fraction of autologous blood having a platelet concentration above baseline suspended in a small amount of plasma Does not include RBCs, theoretically does not include WBCs (although many commercial prep systems include leukocytes) Unclear if other plasma proteins (e.g. albumin, globulins) and circulating small molecules are in plasma portion of PRP (information not available) Platelets Alpha granules: Contain PDGF (3 types), TGF-B (2 types), VEGF, EGF, plt factor 4, IL-1, PDAF, PDEGF, ECGF, IGF and cell adhesion molecules (fibronectin/vitronectin etc) 95% of GFs are secreted within 1 hr of plt activation/degranulation; secretion starts within 10 min; they hang out for 5-10 days after activation Dense granules (aka delta granules): contain serotonin, histamine, dopamine, calcium and adenosine Lambda granules contain lysosomes for clot resorption Marx 2001, Ehrenfest 2009 Marx 2004**, Pietrzak 2005* 1

2 Active Ingredients of PRP Processing options for PRP Single centrifuge process Creates 3 layers (plasma, plt + WBC, RBC) Double centrifuge process Further separates platelets +/- WBCs Many commercial ilprep systems now include ld WBCs Cell-sensitive filtration device (newer, 40% faster, similar platelet concentration to centrifugation) Hall et al 2009* Mehta and Watson, 2008; Ehrenfest et al 2009 Processing pitfalls PRP begins to work with initiation of clot formation which causes platelet activation/degranulation; it is important that clotting does not start prior to injection of PRP In order for proper p degranulation to occur, platelet membranes must remain undamaged (avoid injection or venipuncture w/ small gauge needle) Ehrenfest et al 2009 Marx 2004** 2

3 Platelet and growth factor concentrations Platelet & GF concentrations Pietrzak et al 2005* Hall et al 2009* What factors affect platelet and GF concentrations/effectiveness? Processing technique Different systems result in dramatically different concentrations Amount of platelet activation before and after use Processing which damages platelet membranes or results in partial coagulation prior to use decreases effective number of platelets Patient s blood platelet level (although in 1 study this had no bearing on PRP plt count) Patient factors (e.g. decreased growth factors in patients with DM and with chronic injuries) Which concentration is ideal? Studies using everything from 2x-8.5x plasma concentrations of platelets have been reported Ideal concentration not yet determined, and may vary between different tissue types For wound healing, at least 1 million plt/ul is needed In vitro, dose-response relationship seen between plt concentration and mesenchymal stem cell proliferation/type I collagen production In vitro, greater plt concentration indicated greater levels of PDGF, TFG, and EGF but not IGF and VEGF Overexposure to PRP may yield many cells but limited differentiation of cells into appropriate cell lines Pietrzak 2005* Gandhi et al 2006, Mehta and Watson 2008, Pietrzak 2005*, Lacci and Dardik

4 Unanswered questions Which concentration of PRP is ideal? In which tissues? In which patients? Is PRP pro- or anti-inflammatory? Is this tissue or concentration dependent? Studies have shown both pro- and anti-inflammatory properties; these seem to vary based on tissue type/plt concentration Is PRP with leukocytes also anti-microbial? In vitro studies support this idea What else is in PRP? Are plasma proteins incorportated? What about plasma protein bound substances (e.g. drugs)? Lacci and Dardik 2010, Hall et al 2009*, Crane and Everts 2008** Active ingredients of PRP Transforming Growth Factor-B (TGF-B 1 and 2) Polypeptide that stimulates proiferation of osteoblast precursors and directly stimulates bone collagen synthesis (if injected near bone) Activation of fibroblasts to induce collagen formation (seen in vivo in tendon sheath) Activates endothelial cells for angiogenesis Activates chondroprogenitor cells for cartilage Activates mesenchymal cells Mehta and Watson 2008, Klein et al 2002 Active ingredients of PRP Platelet derived growth factor (PDGF) Dimeric protein with 2 subunits (a and B) that join in different combinatiosn (aa, BB and ab) First growth factor to start nearly all wound healing Stimulates mitogenesis of mesenchymal stem cells and osteoprogenitor cells (when injected near bone) Activates macrophages Stages of Healing Mehta and Watson

5 Inflammatory stage: Clot formation 1 st Tissue Injury: Clotting cascade initiated via Intrinsic (damage to tissue itself) or Extrinsic (exposure to damage tissue) pathway 2 nd Activation: Platelets aggregate 3 rd Degranulation: Alpha granules and dense granules within platelets release protein contents (growth factors and cytokines) 4 th Growth factors attach to receptors (tyrosine kinases) on cell membrane causing intracellular changes Growth factors and Cytokines from Alpha granules Most important are PDGF, TGF β and VEGF Released within 10 min after clotting >95% of presynthesized growth factors are secreted within 1 hour; PLTs secrete and synthesized additional growth factors for remaining life span (2 9 days) Important roll in chemotaxis, cell proliferation, cell differentiation and angiogenesis Important Growth Factors Platelet Derived Growth Factor (PDGF) First growth factor to initiate almost all wound healing Proliferation of osteoblast and fibroblast Stimulates celullar replication/mitogenesis Chemoattractive ti for wbc and mesenchymal stem cells Transforming Growth Factor Beta (TGF β) Stimulates proliferation of osteoblast and fibroblast for collagen formation Increases type I collagen productions in tendon sheath fibroblasts Promotes angiogenesis and cartilage formation Vascular endothelial growth factor (VEGF) 5

6 Bioactive factors from Dense Granules Contain serotonin, histamine, dopamine, calcium and adenosine Histamine and serotonin are released to increase permeability, which allow inflammatory cells greater access to the wound site and activates macrophages Adenosine modulates inflammation/promotes platelet aggregation PRP Administration/Activating Factors Combination with Carbon Chloride and/or thrombin initiates platelet activation, clot formation and growth factor release at the injection site (Gel like consistency) Adding collagen type I has been found to be as effective as thrombin Tendon healing PRP increases in Collagen gene expression and VEGF and HGF de Mos et al (2008): In human tenocytes cultures, PRP stimulates cell proliferation and total collagen production. Also there was increase of matrix degrading enzymes. Mishra et al (2009): Found that 10% buffered inactivated PRP significantly enhances skin fibroblast and mesenchymal proliferation in vitro. Platelets can also be activated by exposure to tendon derived collagen alone 6

7 Tendon healing Greater cell proliferation and angiogenesis in animal tendon models Virchenko et al. (2006) Greater initial regeneration in a rat Achilles tendon defect treated with PRP and greater tendon strength at 14 days PRP may accelerate the initial inflammation phase of tendon repair, making cells more receptive to earlier mechanical loading. Mobilization of circulation derived cells to area of injection and simulates type I collagen production. Muscle healing PRP regulates inflammatory phase Menetrey et al. (2000) Insulin growth factor 1 (IGF 1) and basic fibroblast growth factor were found to improve muscle healing and strength. Hammond et al (2009) An improvement in time to recovery was seen only in the high repetition muscle injury model, suggesting a greater effect of PRP on myogenesis than on sarcolemmal repair. Safety Due to autologous nature, risks are minimal Relative contraindications: History of thrombocytopenia Use of anticougulant therapy Active infection, tumor,metastaticdiseasemetastatic Pregnancy The use of bovine thrombin to activate platelets can lead to formation of antibodies against bovine thrombin which can result in an immunemediated coagulopathy. Further studies required Acutely injured vs degenerative tendon Cellular and molecular process are different Effect of local tissue ph onprp activity Timing of injection to injury Single application vs series of injections Volume injection/application Buffering/activation 7

8 Questions? 8

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