CC BY
16PLASMA RICH IN PLATELETS: CURRENT VIEWS ON THE DEVELOPMENT OF
PREVENTIVE MEDICINE
MD, prof..PavlenkoO.V.
doctor of medicine, professor Institute of Dentistry National Medical Academy of Postgraduate Education named after P.L.Shupyk search engine Bida R.Yu. Institute of Dentistry National Medical Academy of Postgraduate Education named after P.L.Shupyk
ABSTRACT
Tissue engineering and regenerative medicine depend on the relationship between three fundamental elements: progenitor cells such as stem cells, osteoblasts and chondrocytes; signaling molecules such as growth factor, morphogenetic proteins and adhesions and an appropriate structural or carrier basis such collagen, bone or synthetic materials. In other words, most tissues have undifferentiated mesenchymal cells, capable of generating other cells of the same embryonic origin, which depends on the action of modulators that stimulate or inhibit their cellular division, differentiation and migration, as well as their gene expression. In turn, carriers support cell proliferation in the forming tissue as well as transport growth factors and progenitor cells.Recent studies with biomaterials, including various types of platelet concen-trates, have demonstrated the utility of mesenchymal cells (stem cells from bone marrow, fibroblasts, pre-chondrocytes, pre-adipocytes, etc.) for tissue regeneration due to their great potential for self-replication and for replacing the source of tissue- forming cells. Mesenchymal stem cells derived from bone marrow can also play an important role in healing, since they contribute to collagen deposit and support the regeneration of vascular, epithelial and dermal structures.
Biomaterial injection has also been used as a scaffold for cell proliferation and tissue regeneration due to its technical simplicity and non-invasive application procedures. A number of injectable matrices, such as type I collagen, hyaluronic acid, and autologous blood cells are recommended for soft tissues. Fibroblast injections are used based on the hypothesis that autologous fibroblasts are capable of producing collagen without immunologic or allergic reactions. The therapeutic basis of platelet-rich plasma use in medicine is derived from the growth factor content and provisional matrix provided by the platelets themselves. This article briefly reviews the platelet research which led to the conceptual development of PRP as a treatment and also the early history of its use. An overview of platelet structure and function is provided to enhance the clini-cian's understanding of the cell biology behind PRP therapy. The 2 major growth factors in PRP (PDGF and TGFb) are also discussed. Finally, a review of the experimental PRP literature (in vitro and animal studies) is presented, which describes the evidence for use of PRP in tendon/ligament, bone, and joints. Standardization of PRP use remains a challenging prospect due to the number of variables involved in its preparation and administration. It may be that individually-tailored PRP protocols are actually more beneficial for our patients—only time and further research will bear this out.
Keywords: platelet concentrates, plasma rich in platelets, platelet adhesive, growth factors.
For two decades, platelet-rich plasma (PRP) is used in surgery for the treatment of maxillo-facial injuries. PRP contains growth factors and biologically active proteins that help in the treatment of damaged ligaments, muscles and bones. This article highlights the current scientific knowledge about PRP and describes its application in medicine [1^.91;5, р.58;9,р.253;11,р.45;17,р.225;23,р.581;30,р.177].
Research into the biology of bone regeneration, ligaments and muscels have led to the development of a variety of products designed to help stimulate the biological factors and promote healing. The use of autologous and recombinant products quickly increased the possibility of manipulating oral surgery growth factors and secretory proteins, in order to improve the healing of bone and soft tissue. But they are not used in the clinic because many of these products were examined using rigorous scientific standards[3р.491;,6,р.488;13,р.511;23,р.581]. Platelet Rich Plasma (PRP) - is one of such examples. This autologous product was first used and studied in the 70s of the last century. PRP healing properties have been used in clinical practice to increase the concentration of autologous growth factors and secretory proteins, which could enhance regeneration processes at the cellular level [6,р.487;16,р.4;25,р.22]. There is a hope that strengthens PRPrecruitment, proliferation and differentiation of cells involved in tissue regeneration. In the literature, PRP-products, also known as the PRP enriched platelet concentrate
or platelet gel preparation rich in growth factors were investigated in vitro and in vivo experiments in maxillofacial and general surgery [5^.57^0^.302^4^.1033]. Moreover, the role of prosthetic works PRP has been investigated in the process of healing of muscles and bones and it is becoming better known to the general public. The potential of autologous fibrin glue for clinical use was first documented in 1909. It was first introduced in surgical procedures for its sealing properties and to help with homeostasis. Throughout the twentieth century, discoveries were made regarding plateletactivation and the role of growth factors in tissue regeneration [4^.488;8, р.149;18,р.489;21,р.170].
The use of platelet concentrates to substitute fibrin glues has been explored since the 1990s due to the complexity and high costs of producing fibrin concentrates. In 1990, Knighton et al. tested the use of autologous platelets to treat chronic ulcers, with a reduction of almost 50 % in healing time. Similarlyobserved expressive results when using the same technique to treat chronic ulcers in patients for whom limb amputation was initially recommended, with amputation prevented in 78 % of the cases. Such good results made the 1990s a milestone for studies showing the positive action of platelet-derived growth factors.From 1995 to 1997, attempts were made to experimentally confirm the multi-centric therapeutic utilization of growth factors derived from
autologous platelets, their biological safety and techniques for their clinical application to stimulate fibroblastic, endothelial cells [13p.509,17,p.225]. During this period, the osteoinductive and catalyst capacity of fibrin adhesives led to the discovery of their mechanisms of action. Studies also described techniques for usingplatelet gel as an autologous alternative for fibrin glue, which was initially applied in oral surgeries.
Since then, platelet rich plasma (PRP) gradually began to be studied and used in several branches of orthopedic surgery, particularly for perfecting and accelerating healing [17,p.225;20,p.1653;28,p.666].
Platelets are small, non-nuclear elements of the blood, which plays a fundamental role in the hemostatic process. Platelets contain different proteins, cytokines and other bioactive factors that stimulate and regulate the main links healing lesions. The normal number of platelets in the blood is in the range 150,000 - 300,000 per microliter of whole blood. Plasma - a liquid portion of the blood containing the clotting factors and other proteins and ions. Platelet Rich Plasma - a plasma containing about 1 million platelets per microliter of plasma 1. The PRP contained 3-5 times more growth factors than in whole blood. Platelet-enriched plasma can potentially enhance healing due to a variety of growth factors and cytokines secreted from platelet a-granules. Major cytokines found in platelets include transforming growth factor ^ (TGF-^), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), fibroblast growth factor (FGF), epidermal growth factor, a growth factor vascular endothelial (VEGF) and endothelial cell growth factor [1,p.91;2,p.23;3,p.489;4,p.490;13,p.511; 14,p.1040]. These cytokines play an important role in the processes of cell proliferation, chemotaxis, differentiation, and angiogenesis.Hemostasis is the result of the combined action of three main mechanisms: vascular response, platelet activity and blood clotting. When in contact with an injured vascular endothelial surface, even of biological origin, the platelets begin an adhesion reaction to the injury location, releasing pseudopods that facilitate their aggregation, which initiates the hemostatic plug that serves as a base for aggregation factors to affix themselves to the area, which results in the formation of the fibrin network that will obstruct the vascular injury. This process makes the platelets bloated and emit extensions, or pseudopodia, which increase their adhesion capacity and mark the beginning of platelet aggre-gation and the secretion and release of the substances contained in the dense and alpha granules. The release of the calcium ions inside the platelet makes the myofibril within it contract, thus allowing the aggregation and release of the content of the granules. This is serum calcium, which is necessary for the formation of the fibrin network. The presence of the Ca2+ ions in the plasma makes the coagulation factors activate and group, forming the fibrin network, which is sta-bilized by factor XIII and transformed in a stable clot. The calcium ions also inhibit the anticoagulant activity of heparin, preserving the clot [3,p.489;7 ,p.1217;10,p.303;19,p.729;25,p.22].
The presence of thrombin induces the conversion of fibrinogen into fibrin and acts as a platelet activator. After they are activated, the platelets begin to release anti-microbial peptides that help amplify the organism's immune response to the invasion and proliferation of possible infectious agents
in the injured area. Human platelet antimicrobial peptides (HPAPs) are released only in the presence of thrombin, and act basically in two ways: inhibiting or killing pathogens and recruiting a larger quantity of leucocytes and/or lymphocytes to the injured area . Thromboxane A2 then recruits nearby platelets and aggregates them to those that are already activated, continuing the formation of the platelet plug and interrupting bleeding [7,p.1217;12,p.108;15,p.124].The coagulation system involves complex alterations of a set of plasma proteins that participate in the homeostasis process. Its formation begins with the structuring of a fibrin network, which is a protein matrix retaining platelets and red cells that occludes vascular injury. Soon afterwards the clot is retracted, which forces the edges of the injured vessel closer together. Then the clot goes through an organization process, characterized by the invasion of fibroblasts that are attracted by the platelet growth factors, which forms scar tissue. Concomitantly, proteolytic enzymes participate in the clot's dissolution process [11,p.45;18,p.489].
Dietary factors also contain platelets in their dense granules. They contain serotonin, histamine, dopamine, adenosine and calcium ions. These factors are not relevant to growth, but they also play a fundamental role in the healing process. There are 3 stages of healing: inflammation, proliferation, and reconstruction. Inflammatory phase begins immediately after injury, resulting platelets are activated, aggregate and secrete growth factors, cytokines, and haemostatic factors necessary for the early stages of the clotting process. Histamine and serotonin, isolated platelets activate macrophages and increase vascular permeability, which allows access to the site of inflammation. Polymorphonuclear leukocytes migrate to inflammation and shortly thereafter cells begin to proliferate, while fibroblasts help to form a basic substance. After activation of adenosine receptors occurs regulation of inflammation and healing of damage [6,p.488;9,p.251;15,p.124;17,p.225;20,p.1649].
Platelets in PRP participate in the formation of a blood clot, which contains a number of cell adhesion molecules including fibronectin, vitronectin, and fibrin. These molecules play an important role in cell migration and processes of interest in the study of the bioactive properties of the PRP. Thrombus itself may also play a role in the healing of injuries.
Platelet-rich plasma can be prepared only from incoagulated blood. It can not be prepared from clotted whole blood from which the serum is prepared normally, because most of the platelets remain in the formed clot. It is also impossible to prepare PRP from serum, which is a transparent liquid obtained from whole blood clotted and liberated from the cells and proteins involved in the clotting process. Serum contains a very small amount of platelets. Blood, for the preparation of PRP collected in a container containing sodium citrate, which binds calcium ions, thereby blocking the entire coagulation cascade [1,p.91;2,p.17;3,p.490;8,p.137;11,p.45]. This is followed by centrifugation, a step which is carried out in one or two stages. When the plasma is first centrifuged, and platelets are separated from the erythrocytes and leukocytes. Erythrocytes (diameter 7 mm) and leukocytes (diameter 7-15 mm) are much larger and heavier platelets (diameter 2 mm) and are readily separated. A second centrifugation is carried out more smoothly as a result of PRP concentrates; it remains in the supernatant platelet-poor plasma. Next important step is the
activation and aggregation of platelets, leading to the release of biologically active factors contained in the platelets. Some systems for use PRP, commercially available bovine thrombin used as collapses agent. During 10 minutes of platelets released about 70% contained therein bioactive factors, and for 1 hour almost 100%. However, the use of bovine thrombin could result in complications associated with the formation of antibodies against it. This complication is very unlikely, but potentially possible and can lead to such serious diseases such as immunemediated coagulopathy.
Also clots formed by thrombin demonstrate significant retraction. An alternative method for the activation of platelets is the use of "fibrin matrix." Fibrin matrix formed from autologous fibrin which is formed from fibrinogen by the action of its own thrombin formed by adding calcium chloride to the PRP (CaCl2). Calcium chloride is added to a second centrifugation, which results in the formation of dense fibrin matrix. Intact platelets interact with fibrin network formed and activated. This technique is characterized by activation PRP low thrombin formed and thus minimize platelet activation. As a result, the platelets release growth factors are quite slow, and the process can take up to 7 days. A third way is to use the activation PRP type II collagen. It has been shown that collagen is also effective as thrombin stimulates the release of platelet growth factors PDGF and VEGF [2,p.15;5,p.57;9,p.25 6;14,p.1033]. On the other hand, the clumps formed by using collagen exposed retraction not as strong as generated by the action of thrombin.
Connective tissue such as ligaments and muscles to heal in three phases: inflammation, proliferation and reconstruction. Various cytokines are actively involved in all these phases. Cytokines play a major role in wound damage through interaction with transmembrane receptors on the local and circulating cells initiate transmission of intracellular signals that ultimately affects the expression of genes in the nucleus. As a result of the expression of the proteins appear to regulate cell proliferation, cell chemotaxis, angiogenesis, cell differentiation and the formation of extracellular matrix. It is known that cytokines and other bioactive factors extracted from PRP affect basic metabolic processes in the soft tissues [3,p.489;7,p.1217;1 0,p.303;17,p.225;19,p.729;24,p.418].
PRP effect on the muscles. Some cytokines contained in the OTP have a positive effect on the healing of damaged muscle. For example, in gastrocnemius muscle rupture model mice basic fibroblast growth factor (bFGF) and IGF-I to improve muscle healing. Autologous serum is given after 2, 24 or 48 hours after injury in mouse gastrocnemius muscle accelerated cell activation and concomitant increased diameter myofibers regeneration zone [16,p.4;27,p.845;29,p.188].
The literature has accumulated a large number of clinical application of PRP data in areas such as maxillofacial surgery, otolaryngology, plastic surgery and general surgery.
LITERATURE:
1. Anitua E. Autologous platelets as a source of proteins for healing and tissue regeneration / Anitua E., Andia, I., Ardanza, B., et al. // Thromb. Haemost. - 2004. - N. 4. - P. 91.
2. Anitua E. Gel di plasma rico di piastrine / E. Anitua // Implantol. Orale. - 2001. - N. 9. - P. 9-24. 107.
3. Anitua E. The use of plasma-rich in growth factors (PRGF) in oral surgery / E. Anitua // Pract. Proced. Aesthet. Dent. - 2001. - N. 13. - P. 487-493.
4. Anitua E. The use of plasma-rich in growth factors (PRGF) in oral surgery / E. Anitua // Pract. Proced. Aesthet. Dent. - 2001. - N. 13. - P. 487-493.
5. Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg. 2004;33(1):56-9.
6. De Obarrio JJ, Arauz-Dutari JI, Chamberlain JM, Croston A. The use of autologous growth factors in periodontal surgical therapy: platelet gel biotechnology - Case reports. Int J Periodontics Restorative Dent. 2000;20(5):486-97.
7. Different preparation techniques to obtain plateled -rich plasma as source of growth factors for local application/ Zimmerman R,. Jakubietz R,. Schlegel K.A., Wiltfang J. // Tranfusione. - 2001.-Vol. 41. - P.1217
8. Ehrenfest et al. Biology of platelet derived growth factor inSkeletal Growth Factors. Philadelphia: Lippincott Williams & Wilkins, 2000. - P. 129- 151.
9. EG. Evaluation of platelet - rich plasma in combination with freeze-dried bone in the rabbit cranium// Clin. Oral. Impl.
- 2005. - №16. - P.250-257.
10. Fuerst G, Gruber R, Tangl S, Sanroman F, Watzek G. Effects of fibrin sealant protein concentrate with and without platelet-released growth factors on bony healing of cortical mandibular defects. An experimental study in minipigs. Clin Oral Implants Res. 2004;15(3):301-7.
11. Froum, S. J. Effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts: Three bilateral case reports / Froum, S. J., Wallace, S. S., Tarnow, D. P., et al. // Int. J. Periodontics Restorative Dent. - 2002. - N. 22. - P. 45.
12. Kim E. S. Platelet concentration and its effect on bone formation in calvarial defects: an experimental study in rabbits / Kim E. S., Choung P. H. // Med Oral Pathol. - 2010. - P. 108.
13. Kim J. I. Blood vessels of the peri-implant mucosa: a comparasion between the flap and flapless procedures / Kim J. I., Choi B. H., Li J., Xuan F., Jeong S. M. // Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. - 2009. - N. 107(4). - P. 508 -512.
14. Lieberman J. R. The role of growth factors in the repair of bone: biology and clinical applications / Lieberman J. R., Daluiski A., Einhorn Th. A. // Bone joint Sing Am. - 2002.
- N. 84-A. - P. 1032-1044.
15. Man D. The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin glue) in cosmetic surgery / Man D., Plosker H., and Winland-Brown J. E. // Plast. Reconstr. Surg. - 2001. - N. 107. - P. 229.
16. Mann K. G. Thrombin formation / K. G. Mann // Chest. - 2003. - N. 124 (3). - 4 p.
17. Marx R. E. Platelet-rich plasma (PRP): What is PRP and what is not PRP? / R. E. Marx // Implant Dent. - 2001. - N. 10. - P. 225.
18. Marx R. E. Platelet-rich plasma: Evidence to support its use / R. E. Marx // J. Oral Maxillofac. Surg. - 2004. - N. 62. -P. 489.
19. Petrungaro P. S. Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodontal surgery
/ P. S. Petrungaro // Compend. Contin. Educ. Dent. - 2001. -N. 22. - P. 729.
20. Sanchez M. Plasma rich in growth factors to treat an articular cartilage avulsion / Sanchez M., Azofra J., Santisteban J. et al. // Med. Set. Sports. Exer. - 2003. - P. 1648-1653.
21. Siebrecht M. A. Platelet concentrate increases bone ingrowth into porous hydroxyapatite / Siebrecht M.A., De Rooij P.P., Arm D.M., Olsson M.L., Aspenberg P. // Orthopedics. -2002. - N. 25. - P. 169-172.
22. Suba Z. Facilitation of ^-tricalcium phosphate-induced alveolar bone regeneration by platelet-rich plasma in beagle dogs: a histologic and 203 histomorphometric study / Suba Z., Takacs D., Gyulai-Gaal S., Kovacs K. // Int. J. Oral Maxillofac. Implants. - 2004. - N. 19. - P. 832-838.
23. Steed DL. The role of growth factors in wound healing. Surg Clin North Am. 1997;77(3):575-86.
24. Tokuoka K., Hammano H., Ohta T. An adult case of purulent meningitis secondary to retropharyngeal and deep neck abscess after treatment of odontogenic infection // Clin. Neurol. - 1997.-Vol.37, №5. - P. 417-419.
25. Tischler M. Platelet rich plasma: The use of autologous growth factors to enhance bone and soft tissue grafts / M.
Tischler // N. Y. State Dent. J. - 2002. - N. 68. - P. 22.
26. Welsh W. J. Autologous platelet gel: Clinical function and usage in plastic surgery / Welsh W. J. // Cosmetic Derm. -2000. - N. 11. - P. 13. 274.
27. Werner S. Regulation of wound healing by growth factors and cytokines / Werner S., Grose R. // Physiol. Rev. -2003. - N. 83. - P. 835-870.
28. Weibrich G, Hansen T, Kleis W, Buch R, Hitzler WE. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone. 2004;34(4):665-71.
29. Wiltfang J, Kloss FR, Kessler P, Nkenke E, Schultre-Mosgau S, Zimmermann R et al. Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment. Clin Oral Impl Res. 2004;15(2):187-93.
30. Zhu SJ, Choi BH, Jung JH, Lee SH, Huh JY, You TM et al. A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(2):175-9.
