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Mechanism of Platelet Rich Plasma (PRP) Therapy Promoting Tissue Healing

Today, the concept known as PRP first appeared in the field of hematology in the 1970s. Hematologists created the term PRP decades ago to describe the plasma obtained from a platelet count higher than the basic value of peripheral blood. More than ten years later, PRP was used in maxillofacial surgery as a form of platelet rich fibrin (PRF). The content of fibrin in this PRP derivative has important value due to its adhesiveness and steady-state characteristics, while PRP has sustained anti-inflammatory properties and stimulates cell proliferation. Finally, around the 1990s, PRP began to become popular. Finally, this technology was transferred to other medical fields. Since then, this kind of positive biology has been widely studied and applied to the treatment of various musculoskeletal injuries of professional athletes, which further promoted its widespread attention in the media. In addition to being effective in orthopedics and sports medicine, PRP is also used in ophthalmology, gynecology, urology and cardiology, pediatrics and plastic surgery. In recent years, PRP has also been praised by dermatologists for its potential in treating skin ulcers, scar repair, tissue regeneration, skin rejuvenation and even hair loss.

PRP

Considering the fact that PRP can directly manipulate the healing and inflammatory processes, it is necessary to introduce the healing cascade as a reference. The healing process is divided into the following four stages: hemostasis; Inflammation; Cell and matrix proliferation, and finally wound remodeling.

 

Tissue Healing

The tissue healing cascade reaction is activated, which leads to platelet aggregation The formation of clots and the development of temporary extracellular matrix (ECM). Then, platelets adhere to the exposed collagen and ECM protein, triggering the release of bioactive molecules present in a-granules. Platelets contain a variety of bioactive molecules, including growth factors, chemotherapy factors and cytokines, as well as proinflammatory mediators, such as prostaglandin, prostate cyclin, histamine, thromboxane, serotonin and bradykinin.

The final stage of the healing process depends on the remodeling of the wound. Tissue remodeling is strictly regulated to establish a balance between anabolic and catabolic reactions. At this stage, platelet-derived growth factor (PDGF) and transforming growth factor (TGF- β) Fibronectin and fibronectin stimulate the proliferation and migration of fibroblasts, as well as the synthesis of ECM components. However, the time of wound maturation depends to a large extent on the severity of the wound, individual characteristics and the specific healing ability of the injured tissue. Some pathophysiological and metabolic factors can affect the healing process, such as tissue ischemia, hypoxia, infection, growth factor imbalance, and even metabolic syndrome related diseases.

The proinflammatory microenvironment interferes with the healing process. More complicated is that high protease activity inhibits the natural action of growth factor (GF). In addition to its mitotic, angiogenic and chemotactic properties, PRP is also a rich source of many growth factors. These biomolecules may counteract the harmful effects in inflammatory tissues by controlling increased inflammation and establishing anabolic stimuli. Considering these characteristics, researchers may find great potential in treating various complex injuries.

Many diseases, especially those of musculoskeletal nature, strongly depend on biological products that regulate the inflammatory process, such as PRP for the treatment of osteoarthritis. In this case, the health of articular cartilage depends on the precise balance of anabolic and catabolic reactions. With this principle in mind, the use of certain positive biological agents may prove successful in achieving a healthy balance. PRP because it releases platelets α- Growth factors contained in granules are widely used to regulate the potential of tissue transformation, which also reduces pain. In fact, one of the main goals of PRP treatment is to stop the main inflammatory and catabolic microenvironment and promote the transformation to anti-inflammatory drugs. Other authors have previously demonstrated that thrombin activated PRP increases the release of several biological molecules. These factors include hepatocyte growth factor (HGF) and tumor necrosis factor (TNF- α)、 Transforming growth factor beta1 (TGF- β 1) , vascular endothelial growth factor (VEGF) and epidermis Growth factor (EGF). Other studies have shown that PRP promotes the increase of type ii collagen and aggrecan mRNA levels, while reducing the inhibition of the pro-inflammatory cytokine interleukin – (IL) 1 on them. It was also suggested that due to HGF and TNF- α [28] PRP may help to establish anti-inflammatory effect. Both of these molecular preparations reduce the nuclear factor kappaB (NF- κВ) Anti activation activity and expression; Secondly, through TGF- β 1 expression also prevents monocyte chemotaxis, thereby counteracting TNF- α Effect on the transactivation of chemokines. HGF seems to play an indispensable role in the anti-inflammatory effect induced by PRP. This potent anti-inflammatory cytokine destroys NF- κ B signaling pathway and proinflammatory cytokine expression inhibit inflammatory response. In addition, PRP can also reduce the high level of nitric oxide (NO). For example, in articular cartilage, the increase of NO concentration has been proved to inhibit collagen synthesis and induce chondrocyte apoptosis, while increasing the synthesis of matrix metalloproteinases (MMPs), thereby promoting the transformation of catabolism. In terms of cell degeneration, PRP is also considered to be able to manipulate autophagy of specific cell types. When reaching the final aging state, some cell groups lose the potential of static state and self renewal. However, recent studies have shown that PRP treatment can well reverse these harmful conditions. Moussa and colleagues proved that PRP can induce the protection of chondrocytes by increasing autophagy and anti-inflammatory markers, while reducing the apoptosis of human osteoarthritis cartilage. Garcia Pratt et al. It is reported that autophagy determines the transition between the resting and aging fate of muscle stem cells. The researchers believe that, in vivo, the normalization of integrated autophagy avoids the accumulation of intracellular damage and prevents the aging and functional decline of satellite cells. Even in aging human stem cells, such as recently, Parrish and Rodes have also made significant contributions, further revealing the anti-inflammatory potential of PRP. This time, the focus is on the interaction between platelets and neutrophils. In their investigation, the researchers explained that activated platelets released by arachidonic acid were absorbed by neutrophils and converted into leukotrienes and prostaglandins, which are known inflammatory molecules. However, the platelet neutrophil interaction allows leukotriene to be converted into lipoproteins, which have been proven to be an effective anti-inflammatory protein that can limit the activation of neutrophils and prevent dialysis, and promote inheritance to the final stage of the healing cascade.

The proinflammatory microenvironment interferes with the healing process. More complicated is that high protease activity inhibits the natural action of growth factor (GF). In addition to its mitotic, angiogenic and chemotactic properties, PRP is also a rich source of many growth factors. These biomolecules may counteract the harmful effects in inflammatory tissues by controlling increased inflammation and establishing anabolic stimulation.

 

Cell Factor

Cytokines in PRP play a key role in manipulating the process of tissue repair and regulating inflammatory damage. Anti inflammatory cytokines are a wide range of biochemical molecules that mediate the response of proinflammatory cytokines, mainly induced by activated macrophages. Anti inflammatory cytokines interact with specific cytokine inhibitors and soluble cytokine receptors to regulate inflammation. Interleukin (IL) – 1 receptor antagonists, IL-4, IL-10, IL-11 and IL-13 are classified as the main anti-inflammatory drugs, cytokines. According to different wound types, some cytokines, such as interferon, leukemia inhibitory factor, TGF- β And IL-6, which can show proinflammatory or anti-inflammatory effects. TNF- α、 IL-1 and IL-18 have certain cytokine receptors, which may inhibit the proinflammatory effect of other proteins [37]. IL-10 is one of the most effective anti-inflammatory cytokines, which can down regulate proinflammatory cytokines such as IL-1, IL-6 and TNF- α, And up regulate anti-inflammatory factors. These anti regulatory mechanisms play a key role in the production and function of proinflammatory cytokines. In addition, certain cytokines may trigger specific signal responses to stimulate fibroblasts, which are critical for tissue repair. Inflammatory cytokine TGF β 1、IL-1 β、 IL-6, IL-13 and IL-33 stimulate fibroblasts to differentiate into myofibroblasts and improve ECM [38]. In turn, fibroblasts secrete cytokine TGF- β、 IL-1 β、 IL-33, CXC and CC chemokines promote inflammatory response by activating and recruiting immune cells such as macrophages. These inflammatory cells play multiple roles in the wound, mainly by promoting wound clearance – and the biosynthesis of chemokines, metabolites and growth factors, which is crucial for the reconstruction of new tissues. Therefore, cytokines in PRP play an important role in stimulating cell type mediated immune response and promoting the regression of inflammatory stage. In fact, some researchers nominated this process as “regenerative inflammation”, indicating that the inflammatory stage, despite the patient’s anxiety, is a necessary and critical step for the successful conclusion of the tissue repair process, taking into account the epigenetic mechanism that inflammation signals promote cell plasticity.

The role of cytokines in fetal skin inflammation is of great significance to the research of regenerative medicine. The difference between fetal and adult healing mechanisms is that damaged fetal tissues sometimes return to their original state according to fetal age and relevant tissue types. In humans, fetal skin can regenerate completely within 24 weeks, while in adults, wound healing can lead to scar formation. As we have known, compared with healthy tissues, the mechanical properties of scar tissues are significantly reduced, and their functions are limited. Particular attention is paid to cytokine IL-10, which is found to be highly expressed in amniotic fluid and fetal skin, and has been proven to play a role in scar free repair of fetal skin, promoted by the pleiotropic effect of the cytokine. ZgheibC et al. The transplantation of fetal skin into transgenic knockout (KO) IL-10 mice and control mice was studied. IL-10KO mice showed signs of inflammation and scar formation around the grafts, while the grafts in the control group showed no significant changes in biomechanical properties and no scar healing.

The importance of regulating the delicate balance between the expression of anti-inflammatory and pro-inflammatory cytokines is that the latter, when overproduced, ultimately sends signals of cell degradation by reducing the expression of certain genes. For example, in musculoskeletal medicine, IL-1 β Down regulate SOX9, which is responsible for cartilage development. SOX9 produces important transcription factors for cartilage development, regulates type II collagen alpha 1 (Col2A1), and is responsible for encoding type II collagen genes. IL-1 β Finally, the expression of Col2A1 and aggrecan was decreased. However, treatment with platelet rich products has been shown to inhibit IL-1 β It is still a feasible ally of regenerative medicine to maintain the expression of collagen coding genes and reduce the apoptosis of chondrocytes induced by proinflammatory cytokines.

Anabolic stimulation: In addition to regulating the inflammatory state of the damaged tissue, the cytokines in PRP also participate in the anabolic reaction by playing their roles of mitosis, chemical attraction and proliferation. This is an in vitro study led by Cavallo et al. To study the effects of different PRPs on human chondrocytes. The researchers observed that PRP products with relatively low platelet and leukocyte concentrations stimulate normal chondrocyte activity, which is conducive to promoting some cellular mechanisms of anabolic response. For example, the expression of type ii collagen and aggregating glycans was observed. In contrast, high concentrations of platelets and leukocytes seem to stimulate other cellular signaling pathways involving various cytokines. The authors suggest that this may be due to the presence of a large number of white blood cells in this particular PRP formulation. These cells appear to be responsible for increased expression of certain growth factors, such as VEGF, FGF-b, and interleukins IL-1b and IL-6, which may in turn stimulate TIMP-1 and IL-10. In other words, compared with the “bad” PRP formula, the PRP mixture rich in platelets and white blood cells seems to promote the relative invasiveness of chondrocytes.

A study designed by Schnabel et al. was designed to evaluate the role of autologous biomaterials in horse tendon tissue. The authors collected blood and tendon samples from six young adult horses (2-4 years old), and focused on the study of gene expression pattern, DNA and collagen content of the tendon explants of flexor digitorum superficialis of horses cultured in the medium containing PRP or other blood products. Tendon explants were cultured in blood, plasma, PRP, platelet deficient plasma (PPP) or bone marrow aspirates (BMA), and amino acids were added to 100%, 50% or 10% serum free DMEM. In running the applicable biochemical analysis after…, the researchers noted that TGF- β The concentration of PDGF-BB and PDGF-1 in PRP medium was especially higher than that of all other blood products tested. In addition, tendon tissues cultured in 100% PRP medium showed increased gene expression of matrix proteins COL1A1, COL3A1 and COMP, but did not increase catabolic enzymes MMPs3 and 13. At least in terms of tendon structure, this in vivo study supports the use of autolo – a gouty blood product, or PRP, for the treatment of large mammalian tendinitis.

Chen et al. The reconstructive effect of PRP was further discussed. In their previous series of studies, the researchers proved that, in addition to enhancing cartilage formation, PRP also promoted the increase of ECM synthesis and inhibited the inflammatory reaction of articular cartilage and nucleus pulposus. PRP can activate TGF through phosphorylation of Smad2/3- β Signal pathway plays an important role in cell growth and differentiation. In addition, it is also believed that fibrin clots formed after PRP activation provide a solid three-dimensional structure, enabling cells to adhere, which may lead to the construction of new tissues.

Other researchers have made significant contributions to the treatment of chronic skin ulcers in the field of dermatology. This is also noteworthy. For example, the research conducted by Hessler and Shyam in 2019 shows that PRP is of value as a feasible and effective alternative treatment, while drug-resistant chronic ulcer still brings significant economic burden to health care. In particular, diabetes foot ulcer is a well-known major health problem, which makes limbs easy to be amputated. A study published by Ahmed et al. in 2017 showed that autologous PRP gel could stimulate wound healing in patients with chronic diabetes foot ulcer by releasing necessary growth factors, thereby significantly improving the healing rate. Similarly, Gonchar and colleagues reviewed and discussed the regenerative potential of PRP and growth factor cocktails in improving the treatment of diabetes foot ulcers. The researchers proposed that the use of growth factor mixtures is likely to be a possible solution, which can improve the advantages of using PRP and single growth factor. Therefore, compared with the use of single growth factor, the combination of PRP and other treatment strategies may significantly promote the healing of chronic ulcers.

 

Fibrin

Platelets carry several factors related to fibrinolytic system, which may up regulate or down regulate fibrinolytic reaction. The time relationship and relative contribution of hematological components and platelet function in clot degradation is still a problem worthy of extensive discussion in the community. The literature introduces many studies that only focus on platelets, which are famous for their ability to affect the healing process. Despite a large number of outstanding studies, other hematological components, such as coagulation factors and fibrinolytic systems, have also been found to contribute significantly to effective wound repair. By definition, fibrinolysis is a complex biological process that depends on the activation of certain enzymes to promote the degradation of fibrin. Fibrinolysis reaction has been proposed by other authors that fibrin degradation products (fdp) may actually be molecular agents responsible for stimulating tissue repair. The sequence of important biological events before is from fibrin deposition and removal of angiogenesis, which is necessary for wound healing. The formation of clots after injury serves as a protective layer to protect tissues from blood loss and invasion of microbial agents, and also provides a temporary matrix through which cells can migrate during the repair process. The clot is due to fibrinogen being cleaved by serine protease, and platelets are gathered in the cross-linked fibrin fiber mesh. This reaction triggered the polymerization of fibrin monomer, which is the main event of blood clot formation. The clot can also be used as a reservoir of cytokines and growth factors, which are released during the degranulation of activated platelets. The fibrinolytic system is strictly regulated by plasmin, and plays a key role in promoting cell migration, the bioavailability of growth factors and the regulation of other protease systems involved in tissue inflammation and regeneration. The key components of fibrinolysis, such as urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are known to be expressed in mesenchymal stem cells (MSCs), which are special cell types necessary for successful wound healing.

 

Cell Migration

Activation of plasminogen through uPA uPAR association is a process that promotes the migration of inflammatory cells because it enhances extracellular proteolysis. Due to the lack of transmembrane and intracellular domains, uPAR needs co receptors such as integrin and vitellin to regulate cell migration. It further indicated that the binding of uPA uPAR resulted in an increase in the affinity of uPAR for vitrectonectin and integrin, which promoted cell adhesion. Plasminogen activator inhibitor-1 (PAI-1) in turn makes cells detach. When it binds to uPA of uPA upar integrin complex on the cell surface, it destroys the interaction between upar vitellin and integrin vitellin.

In the context of regenerative medicine, bone marrow mesenchymal stem cells are mobilized from the bone marrow in the case of severe organ damage, so they may be found in the circulation of patients with multiple fractures. However, in specific cases, such as end-stage renal failure, end-stage liver failure, or during rejection after heart transplantation, these cells may not be detected in the blood [66]. Interestingly, these human bone marrow derived mesenchymal (stromal) progenitor cells could not be detected in the blood of healthy individuals [67]. The role of uPAR in the mobilization of bone marrow mesenchymal stem cells (BMSCs) has been proposed previously, which is similar to the occurrence of uPAR in the mobilization of hematopoietic stem cells (HSCs). Varabaneni et al. The results showed that the use of granulocyte colony-stimulating factor in uPAR deficient mice caused MSC failure, which once again strengthened the supporting role of fibrinolysis system in cell migration. Further studies also showed that glycosyl phosphatidylinositol anchored uPA receptors regulate adhesion, migration, proliferation and differentiation by activating certain intracellular signaling pathways, as follows: survivable phosphatidylinositol 4,5-diphosphate 3-kinase/Akt and ERK1/2 signaling pathways, and adhesion kinase (FAK).

In the context of MSC wound healing, fibrinolytic factor has proved its further importance. For example, plasminogen deficient mice showed a severe delay in wound healing events, indicating that plasmin was important in this process. In humans, loss of plasmin can also lead to complications of wound healing. Interruption of blood flow can significantly inhibit tissue regeneration, which also explains why these regeneration processes are more challenging in patients with diabetes.

Bone marrow mesenchymal stem cells were recruited to the wound site to accelerate wound healing. Under stable conditions, these cells expressed uPAuPAR and PAI-1. The last two proteins are hypoxia inducible factors α (HIF-1 α) Targeting is very convenient because HIF-1 in MSCs α The activation of FGF-2 and HGF promoted the up regulation of FGF-2 and HGF; HIF-2 α In turn, VEGF-A [77] is up-regulated, which together contributes to wound healing,. In addition, HGF seems to enhance the recruitment of bone marrow mesenchymal stem cells into wound sites in a synergistic manner. It must be noted that ischemic and hypoxic conditions have been shown to significantly interfere with wound repair. Although BMSCs tend to live in tissues that provide low oxygen levels, the survival of transplanted BMSCs in vivo becomes limited because transplanted cells often die under adverse conditions observed in damaged tissues. The fate of adhesion and survival of bone marrow mesenchymal stem cells under hypoxia depends on the fibrinolytic factors secreted by these cells. PAI-1 has a high affinity for vitellin, so it can compete for the binding of uPAR and integrin to vitellin, thereby inhibiting cell adhesion and migration.

PRF

Monocyte and Regeneration System

According to the literature, there are many discussions about the role of monocytes in wound healing. Macrophages mainly come from blood monocytes and play an important role in regenerative medicine [81]. Because neutrophils secrete IL-4, IL-1, IL-6 and TNF- α, These cells usually penetrate the wound about 24-48 hours after injury. Platelets release thrombin and platelet factor 4 (PF4), which can promote the recruitment of monocytes and differentiate into macrophages and dendritic cells. A significant feature of macrophages is their plasticity, that is, they can convert phenotypes and differentiate into other cell types, such as endothelial cells, and then show different functions to different biochemical stimuli in the wound microenvironment. The inflammatory cells express two major phenotypes, M1 or M2, depending on the local molecular signal as a source of stimulation. M1 macrophages are induced by microbial agents, so they have more proinflammatory effects. In contrast, M2 macrophages are usually produced by type 2 reactions and have anti-inflammatory properties, typically characterized by an increase in IL-4, IL-5, IL-9, and IL-13. It is also involved in tissue repair through the production of growth factors. The transition from M1 to M2 subtype is largely driven by the late stage of wound healing. M1 macrophages trigger neutrophil apoptosis and initiate the clearance of these cells). The phagocytosis of neutrophils activates a series of events, in which the production of cytokines is turned off, polarizing macrophages and releasing TGF- β 1。 This growth factor is a key regulator of myofibroblast differentiation and wound contraction, which allows the resolution of inflammation and the initiation of the proliferation phase in the healing cascade [57]. Another highly related protein involved in cellular processes is serine (SG). This hemopoietic cell secretory granule proteoglycan has been found to be necessary to store secretory proteins in specific immune cells, such as mast cells, neutrophils and cytotoxic T lymphocytes. Although many non hematopoietic cells also synthesize plasminogen, all inflammatory cells produce a large amount of this protein and store it in granules for further interaction with other inflammatory mediators, including proteases, cytokines, chemokines and growth factors. The negatively charged glycosaminoglycan (GAG) chains in SG seem to be critical to the stability of secretory granules, as they can bind to and facilitate the storage of essentially charged granular components in a cell, protein, and GAG chain specific manner. Regarding their participation in PRP research, Woulfe and colleagues have previously shown that SG deficiency is closely related to platelet morphological changes; Platelet factor 4 β- Defects of PDGF storage in thromboglobulin and platelets; Poor platelet aggregation and secretion in vitro and thrombosis defect in vivo. The researchers therefore concluded that this proteoglycan seems to be the main regulator of thrombosis.

Fibrinolytic

Platelet rich products can obtain personal whole blood through collection and centrifugation, and divide the mixture into different layers containing plasma, platelets, white blood cells and white blood cells. When the platelet concentration is higher than the basic value, it can accelerate the growth of bone and soft tissue, with the least side effects. The application of autologous PRP products is a relatively new biotechnology, which has continuously shown optimistic results in stimulating and enhancing healing of various tissue injuries. The efficacy of this alternative treatment method may be attributed to the local delivery of a wide range of growth factors and proteins to simulate and support the physiological wound healing and tissue repair process. In addition, fibrinolytic system obviously has an important influence on the whole tissue repair. In addition to changing the cell recruitment of inflammatory cells and bone marrow mesenchymal stem cells, it can also regulate the proteolytic activity of wound healing areas and the regeneration process of mesodermal tissues, including bone, cartilage and muscle, so it is a key component of musculoskeletal medicine.

Accelerated healing is the goal highly pursued by many professionals in the medical field. PRP represents a positive biological tool, which continues to provide promising development in stimulating and coordinating the cascade of regenerative events. However, because this therapeutic tool is still very complex, especially because it releases countless bioactive factors and their various interaction mechanisms and signal transduction effects, further research is needed.

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Post time: Dec-16-2022