Technologies for producing platelet masses for regenerative medicine

Рукопис отримано: 11.10.2019р. Прийнято: 10.11.2019р. Затверджено до друку: 17.12.2019р. The development of regenerative medicine is to improve existing and to search for new tools for morphological and functional tissue repair, among which plasma or fi brin enriched with platelets (PRP and PRF) can be signifi cant. Autogenic platelet masses stimulate collagen synthesis, induce vascular growth, reduce pain, provide hemostasis, accelerate regeneration, reduce the risk of postoperative infectious and infl ammatory complications, and also have powerful osteoinductive properties. Due to the ability to produce the majority of growth factors, platelets can aff ect all stages of the infl ammatory-regenerative process, and therefore their biological products are of great importance in solving the problems of regenerative medicine. The technologies for obtaining PRP and PRF are based on centrifugation of blood, as a result of which its active components are concentrated in certain areas of the centrifuge. Blood sampling with or without an anticoagulant, as well as modifi cation of centrifugation protocols, allows to obtain various forms of platelet masses, such as a liquid, gel or clots. They are classifi ed, depending on the cellular content and architecture of fi brin, into several categories, namely: pure plasma enriched in platelets (P-PRP), plasma enriched in leukocytes and platelets (L-PRP); injectable fi brin enriched with platelets (i-PRF) and pure fi brin enriched with platelets (P-PRF), as well as fi brin enriched with white blood cells and platelets (L-PRF). The main diff erence in the manufacture of PRP compared to PRF is the use of anticoagulants and activators, as well as the possibility of using two-stage centrifugation. Platelet mass is used as an independent component mainly to stimulate the restoration of muscle tissue, to heal chronic wounds, to treat articular pathologies, and in combination with other materials, in particular to replace bone defects. The mechanisms of infl uence of each of the categories of platelet mass on tissue regeneration remains poorly understood. It is necessary to standardize the protocols for their preparation, taking into account the infl uence of additional substances, such as platelet activators or blood clotting and anticoagulants, as well as optimization of the methods for using each of the platelet mass forms.

Problem statement, analysis of recent research. In recent years, regenerative biomedicine has acquired signifi cant theoretical and practical development, which represents scientifi cally based concepts, methods and technologies for the obtaining and storage of cellular and tissueengineered products, restoration and controlled regeneration of tissues and organs, their structures and functions. The problems of regenerative biomedicine are extremely wide. On the one hand, it is caused by signifi cant diff erences in the regenerative properties of tissues and organs, and on the other hand, by the loss of their reparative potential [1,2].
The reasons for this can be various factors, in particular tissue degeneration, oxidative damage and an imbalance of regenerative mechanisms based on the loss of blood supply or large-volume injuries, their complications by infectious and infl ammatory processes, metabolic syndrome or immunopathological processes leading to tissue destruction or metaplasia [3].
This necessitates the improvement and search for new accessible, safe and optimal materials that must meet certain requirements [10]: biocompatibility, stimulation of angiogenesis [5], osteo-conductivity and osteo-inductivity [11], as well as the absence of infl ammatory, allergic and toxic reactions [10].
At the same time, signifi cant attention is paid to various growth factors and bone morphogenetic proteins [12,13]. However, platelet masses can be their alternative; they can enhance and optimize regeneration processes due to the content of all known growth factors in platelets [4,9,14].
Platelets and platelet mass. Platelets are nuclear-free spherical cells with a diameter of 2-4 microns [15]. In the blood stream, they circulate for about 9-11 days, capable of instant adhesion, aggregation, and secretion of their granules contents [16], the fi rst to accumulate in large numbers in areas of damaged vessel and surrounding tissues [17]. These cells contain almost all possible sources of reactive oxygen species, such as xanthine oxidase, cytoplasmic NAD (F) H oxidase, mitochondria, and enzymes that catalyze the conversion of arachidonic acid. Active forms of oxygen perform many functions in the body: participation in the reactions of oxidative phosphorylation, biosynthesis of prostaglandins and nucleic acids, in the processes of mitosis and decay of phagocytized bacterial cells [18].
In platelets there are about 35 α-granules and 5 dense bodies that serve as the main storage tanks for various biologically active substances [17].
Platelet activation occurs by a number of stimulants (thrombin, calcium chloride, collagen, etc.) [19] due to contact with specifi c receptors located on their surface, or as a result of interaction with collagen, von Willebrand factor and other adhesive proteins. At the same time, the intracellular concentration of calcium ions increases, and as a result, the proteins of the platelets cell membrane mediate adhesion and aggregation [15,20,21].
Platelet concentrates are becoming more widely used in various fi elds of humane medicine such as orthopedics, otorhinolaryngology, gynecology, cosmetic surgery, ophthalmology, general surgery and dentistry [22,28], as well as recently in veterinary medicine. Plasma enriched with platelets has powerful osteoinductive properties, and therefore it is combined with various osteoconductive materials [29].
Diff erent forms of platelet mass are made by modifying the protocols of blood centrifuging. Their classifi cation is based on two main parameters, such as fi brin architecture and cell content. Depending on this, platelet concentrates can be divided into 5 main categories [22,33]: -pure platelet-rich plasma (Pure Platelet-Rich Plasma (P-PRP)) or plasma rich in growth factors (Plasma Rich in Growth Factors (PRGF)) -plasma enriched in white blood cells and platelets (Leukocyte-and Platelet-Rich Plasma (L-PRP)) -injectable platelet-rich fi brin (injectable-Platelet-Rich Fibrin (i-PRF)) -pure fi brin enriched with platelets (Pure Platelet-Rich Fibrin (P-PRF)), commercial name Fibrinet (its manufacturing technology involves the use of an anticoagulant) -fi brin enriched with leukocytes and platelets (Platelet-Rich Fibrin (L-PRF)) [37,38,30].
Their molecules induce and regulate angiogenesis, extracellular matrix remodeling, and cellular eff ects: stem cell involvement, chemotaxis, proliferation, and diff erentiation [43]. That is, they are able to infl uence any stage of the regenerative process -infl ammatory, proliferative and remodeling. However, the eff ectiveness of their infl uence on these biological mechanisms largely depends on the degree of release and activity of growth factors and other substances of platelets, which is determined by the technology and protocol of their concentration [28].
These forms of platelet mass are used: liquid, gel [35], clots or fi lms (membranes) of fi brin [44,30], topically, as an injection [45, 46,32], or in combination with other materials [47,29]. A variety of forms and methods of using PRP allows their use for the regeneration of various types of tissues. In this case, the type of centrifuge, its rotation speed [46,48,49] and rotor diameters play an important role, since platelets of diff erent composition and properties are obtained for various parameters, which is the most controversial today [50]. The calculation of the relative centrifugal force (g) allows to determine the desired centrifugation speed for each centrifuge [51].
The following forms of platelet-rich plasma are used: liquid -not activated and gelactivated [42,53,41,49,54]. For the concentration of platelets, one-or two-stage centrifugation is used [55,56]. Moreover, the composition of PRP, depending on the technique, can be signifi cantly diff erent in the content of cells, growth factors and cytokines. It is also aff ected by the method of platelet activation, which aff ects the clinical effi cacy [40,57]. This often leads to a contradictory interpretation of its results [42].
The properties of autologous plateletrich plasma are also aff ected by the rate of capture and the volume of whole blood (which determines the number of platelets in the fi nal plasma volume), the choice of anticoagulant [46,42] (sodium citrate, heparin, sodium EDTA and potassium EDTA (sodium and potassium ethylenediaminetetraacetic acid, which is not recommended for preparation, reduces the loss of grain size)) and pH (7.2 -8.0) [58].
at the base of the tube [51]. After centrifugation, separated from the platelet-depleted plasma, the platelet mass is collected in a syringe and activated using various activators (calcium chloride, calcium gluconate, thrombin). The fi nal product is a gel [42].

Platelet-rich fi brin.
Platelet-rich fi brin contains an autologous fi brin matrix enriched in white blood cells, platelets, and cytokines. Its tetramolecular structure acts as a matrix and is capable of biological resorption. It induces the development of the vasculature and directed cell migration [8]. If a normal clot formed from native blood without centrifugation contains 95% red blood cells, 5% platelets, less than 1% leukocytes and numerous fi brin fi bers, then the platelet concentration enriched with platelets reaches 95% [61].
The advantages of PRF over other categories of platelet mass include ease of preparation, Two-stage centrifugation. In the fi rst stage, red blood cells are separated from plasma and white blood cells with platelets. During the second stage, the fi nal separation of plasma, white blood cells and platelets occurs. As in the previous case, the blood is taken with an anticoagulant and centrifuged. The erythrocyte layer is separated, over which a white blood cell fi lm is formed, and a plasma layer above it. The whole plasma with or without a white blood cell fi lm (depending on what type of plasma needs to be made: pure plasma enriched in platelets or plasma enriched in white blood cells and platelets) is taken with a pipette into another tube, which is subjected to the following centrifugation ( Table 1). As a result, a layer of plasma enriched in platelets is formed the absence of anticoagulants and activators, uniformity and stability [25]. This autologous product does not contain any foreign substances that can adversely aff ect the regeneration processes and is absolutely physiological [8]. Platelet-rich fi brin is used in the form of a clot (PRF) [54,30] and in liquid injection form (i-PRF) [45, 61, 32]. The method of preparation of fi brin enriched in platelets is presented in table. 2. It includes the selection of blood in a plastic tube without an anticoagulant and immediate centrifugation. After this, erythrocytes are concentrated in the lower part of the test tube, a fi brin clot enriched with platelets -in the middle, and serum in the upper part. It should be noted that, as in a platelet-rich plasma, a high concentration of cellular elements in a fi brin clot is located in its lower third. In the upper third, platelets can be found in very small amounts or they are absent [62,63,64]. The most important parameter for the success of this procedure is the minimum possible time interval between the process of blood selection and centrifugation [23,30].
The preparation of i-PRF is based on centrifugation at low speed and the use of plastic tubes to prevent premature blood coagulation.
As a result, blood is distributed into the following fractions: in the lower -erythrocytes, in the middle -red liquid, liquid fi brin enriched in platelets, in which leukocytes and platelets are more concentrated, and in the upper fractionyellow, with fewer cellular elements [32] (table 3). After a few minutes, fi brin polymerizes and turns into a dense clot. The complexity of preparation of injectable fi brin is justifi ed by the wider possibilities of its use in contrast to the PRF clot: in the form of an injection and in combination with other materials, forming a homogeneous composite [45, 46,61]. However, the properties of i-PRF require detailed study [32].
The use of platelet masses. Platelet-rich plasma is able to restore muscle tissue [26], accelerates the healing of skin wounds [67] and chronic wounds [49], reduces synovial edema, joint stiff ness, which indicates its antiinfl ammatory and regenerative properties [68]. The positive eff ect of platelet-rich plasma on tendon repair is also reported [26]. The use of platelet-rich plasma in combination with implants provides restoration of bone tissue defects [47]. Its combination withhydroxyapatite materials improved the regeneration of skull bones, as it was in studies on rats [29], but in some studies there were no particular advantages observed when using platelet-rich plasma [28].
Platelet-rich fi brin was used to heal wounds of the distal limbs [54]. It causes the formation of a large number of blood vessels [69], promotes the restoration of periodontal defects -a study on rats [65], and tissue repair in depulped teeth in dogs [70].
Conclusions. So, platelet rich plasma and fi brin are safe, aff ordable, and biocompatible materials. They can be used both independently and in combination with various components. However, the eff ect of each of the categories of platelet mass on tissue regeneration remains debatable.
The ability of various forms of platelet masses to restore tissue due to the release of growth factors remains insuffi ciently studied. It is necessary to standardize the protocols for the production and classifi cation of these autologous products. As a result of any slightest deviation from the procedure, a biologically active substance with other properties is formed.
Copyright: © Sevchenko S., Rublenko M., Bonkovsky O. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.