Platelet rich plasma (PRP) is a platelet concentrate obtained by gradient centrifugation of autologous whole blood, which is rich in platelets. When platelets are activated, they release a variety of growth factors that play an important role in promoting the proliferation, growth, differentiation and tissue formation of osteocytes and osteoblasts. Since Marx et al. first used PRP composite graft bone to repair mandibular defects in 1998, PRP has been gradually applied in tissue repair in the fields of dentistry, plastic surgery, orthopedics, otolaryngology, and neurosurgery. In this paper, we review the isolation and preparation of PRP and its application in the field of orthopedics, as well as the problems and prospects of its application. 1. Separation and preparation of PRP PRP is a PLT concentrate that is separated from whole blood by density gradient centrifugation according to the different sedimentation coefficients of the constituents in blood. The number of PLT, concentration of various growth factors, and number of leukocytes in PRP prepared by different number of centrifuges, centrifugal force, centrifugal time, and different activation methods of PLT vary; and the biological effects of various surgical procedures and the time of application of PRP also produce different biological effects, thus generating differences in the biological effects of PRP. The preparation of PRP with different growth factor contents according to different physiopathological needs is the direction of future research. The preparation methods of PRP can be broadly divided into two types: manual preparation and fully automated preparation. The manual preparation process is tedious, but the required equipment is simple and easy to carry out. Fully automated preparation requires special equipment, and currently the commonly used equipment includes SmartPReP system, Trissee system, Platele concentrate collection system, Curasau system, etc. There is no significant difference in the number of platelets between the manual separation method and the automatic platelet separator method after centrifugation, although the automatic platelet separator is easy to operate and highly automated, and the purity and concentration of the prepared PRP platelets are high, but this method is generally used for a large amount of blood (generally above 150 ml) or the need to establish a venous circulation channel, and is currently mainly used for platelet collection in blood banks to Currently, it is mainly used for platelet collection in blood banks for component transfusion, which limits its wide application in clinical practice due to its high cost. After one centrifugation, the blood can be divided into three layers, the bottom layer is the red blood cells with the largest settling coefficient, the top layer is the supernatant, and there is a thin layer at the junction, i.e. platelet-rich layer. After one centrifugation, the supernatant or red blood cell layer is discarded, and then the centrifugation force is changed again to separate more platelets. The two-centrifugation method is still the common method for the preparation of PRP. Liu Caixia et al. compared the effects of PRP prepared by different centrifugation force and centrifugation time on distraction osteogenesis in an animal model, and the results showed that the platelet count of PRP prepared by the Landesberg method with two centrifugations (200 × g and 10 min each) was significantly higher than that of whole blood, which was 6.17 times higher than that of whole blood; the platelet recovery rate was more than 86%, and the effect of promoting new bone production was more obvious. The platelet recovery rate was more than 86%, and the effect of promoting new bone production was more obvious. The platelet concentration of PRP obtained by centrifugation at a time < 5 min was not significantly different from that of whole blood when the centrifugation force was > 250 × g. Marx et al. found that platelet concentration was highest in the erythrocyte layer 2 mm below the interface after one centrifugation, and the supernatant was discarded and centrifuged again at low speed for better platelet extraction. However, most scholars believe that the platelet recovery rate is higher by using the modified Appel method, i.e., first centrifugation at low speed and then aspiration of the entire supernatant and a small portion of red blood cells under the junction layer in another centrifuge tube, followed by high speed centrifugation. 2.The mechanism of action of platelet-rich plasma The action of PRP is accomplished through the interaction and mutual regulation of growth factors, which are secreted and immediately adhere to the surface of the target cell membrane and activate the cell membrane receptors. These membrane receptors in turn induce intrinsic signaling proteins that stimulate the normal gene sequence expression of the cell. Thus, the growth factors released by PRP do not enter the target cells and do not lead to changes in the genetic properties of the target cells, but only to an acceleration of the normal healing process. Although the mechanism of action of all cytokines involved in tissue repair and reconstruction is still unclear, some of the effects of cytokines on tissue repair and reconstruction have been clarified, such as: PDGF, one of the first growth factors to appear at the fracture site, can stimulate the mitosis of bone marrow stromal cells and increase the number of osteoblasts; stimulate the growth of endothelial cells and promote capillary production in the recipient area; also stimulate It can also stimulate the chemotaxis of monocyte macrophages. As a mitogenic and biotropic factor, TGF-β is highly expressed in traumatic bone tissue, promoting osteoblast chemotaxis and proliferation, and increasing collagen synthesis. It stimulates the chemotaxis, mitosis and collagen fiber synthesis of osteoblast and osteoclast, inhibits osteoclast formation and bone resorption, and plays an important role in fracture repair; IGF promotes the proliferation and migration of osteoblasts and enhances osteoclast viability; VEGF induces the proliferation and migration of endothelial cells and thus promotes neovascularization. In addition, the activated PLT also releases a large number of proteins, which are important for tissue regeneration. Thrombin can recruit endothelial cells from surrounding tissues and enhance their viability. Under human umbilical vein three-dimensional culture conditions, thrombin can stimulate fibroblast proliferation and neocapillary formation, while inducing negative feedback, thus limiting neocapillary synthesis. Fibronectin stimulates migration of keratinized cells and achieves cell-cell interaction, which is important for cell morphology recovery. PRP is completely autologous, free of disease transmission and immune rejection, which fundamentally solves the problem of disease transmission and the difficulty of graft survival in bone tissue engineering; PRP contains a variety of growth factors in high concentration, and the ratio of each growth factor is similar to the normal ratio in the body, and has the best synergistic effect. The PRP contains high concentrations of multiple growth factors in ratios similar to the normal ratios in vivo, with optimal synergy between the biological effects of a single factor and the interactions of various growth factors. The growth factors contained in PRP do not enter into the cell or nucleus, which accelerates the normal healing process and has no teratogenic effect or ability to induce tumor formation; PRP is easy to make and less damaging to patients, as it can be made by simply taking blood from patients’ veins. Therefore, PRP is a safe, simple, and inexpensive procedure that can be used in various fields of orthopedic treatment and has a wide range of applications. However, there are still many unsolved problems in the clinical use of PRP, especially in the field of orthopedics, such as the lack of uniform standards for PRP preparation, the large differences in the concentration of PRP growth factors prepared by different methods, the number of growth factors contained in PRP and the mechanism of their interaction are still unclear. Therefore, the research on PRP should firstly establish a set of PRP preparation methods with high efficiency and stability, low damage to PLT, high purity and stability; secondly, avoid the factors affecting the efficacy of PRP when applying PRP; select appropriate carriers to bind PRP to carriers to improve the bone regeneration ability of PRP, establish animal models, and design standardized tests to provide a basis for the clinical application of PRP.
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