Chronic nonhealing wounds are not yet clearly defined, but are usually understood as wounds that do not heal through the normal wound healing process due to various intrinsic or external factors and enter a state of pathological inflammatory response, resulting in a persistent wound healing [1]. However, the local application of growth factors is influenced by the microenvironment of the wound surface, which makes it difficult to achieve the expected therapeutic effect due to its rapid inactivation and short duration of action, and is also limited by the route of administration, dosage form and economic burden.
Since the 1990s, some scholars at home and abroad found that platelet-rich plasma (PRP) contains high concentrations of growth factors, and then some scholars found that PRP has clear effects on promoting wound healing, osteogenesis and soft tissue repair and accelerating bone healing, which can significantly shorten wound healing time and improve bone healing quality, and PRP has a good clinical application prospect because it is completely autologous, free from disease transmission and immune rejection, simple to make, and has little damage to tissues [2]. In this paper, we mainly review the preparation, composition, application and prospects of PRP.
1. Platelet-rich plasma
Harke et al [1] first isolated and prepared platelet?rich plasma (PRP) in 1977, and successfully used it in cardiac surgery patients to avoid platelet function damage and postoperative blood loss during cardiopulmonary bypass (CPB). In recent years, autologous PRP has been widely used in cardiac surgery, oral and maxillofacial surgery, and repair of bone and soft tissue defects, with satisfactory results.
1.1 Principle of platelet-rich plasma isolation
1.1.1 The current methods of PRP preparation include plasma separation and replacement and centrifugal separation. Plasma separation and replacement method is to use multi-functional medical blood component automatic separation equipment to collect platelet components alone, with high degree of automation and high purity and concentration of the prepared PRP platelets, but this method is generally used for those who use more blood (generally above 150 ml) or need to establish a venous circulation channel to collect platelets and then transfuse other blood components back. The equipment is expensive, which limits the widespread use in clinical practice, and is currently mainly used for platelet collection in blood banks for component transfusion.
Centrifugal separation method for the preparation of PRP requires low equipment requirements and simple steps, and its production principle is: the settling coefficient of each component in the blood is different, and the blood is divided into three layers after one centrifugation, the bottom layer is the largest settling coefficient of red blood cells layer, the uppermost layer is the serum layer, and there is a thin layer at the junction (not easily visible to the naked eye), that is, the platelet-rich layer; after one centrifugation, the supernatant layer or red blood cells layer is discarded, and then the centrifugal force is changed and centrifuged again to make more platelets separated. Then the centrifugation force is changed again, so that more platelets are separated out. The centrifugation method is usually performed with a lower centrifugal force for the first centrifugation to avoid rapid platelet sedimentation, and a higher centrifugal force for the second centrifugation to promote complete platelet sedimentation in a shorter period of time. It was demonstrated that platelets were most abundant near the interface, and preserving the red blood cell layer lmm below the interface could greatly improve platelet acquisition rate and reduce platelet depletion.
Landesberg et al [2] found that centrifugation with different centrifugal forces and times resulted in excessive platelet destruction, while the platelet concentration of PRP obtained with 1 centrifugation time <5 min was not significantly different from that of whole blood; it is recommended that the red cell layer be discarded after 1 centrifugation, and then centrifuged again with a centrifugal force of 200 g and 10 min for both centrifugations. Marx et al [3] found that the platelet concentration was highest in the erythrocyte layer 2 mm below the interface after 1 centrifugation at high speed, and the supernatant was discarded and centrifuged again at low speed for better platelet extraction. However, most scholars believe that the platelet recovery is higher by using the modified Appel method [4], which involves centrifugation at low speed followed by aspiration of the entire supernatant and a small portion of red blood cells below the junction layer in another centrifuge tube, and then centrifugation at high speed.
1.1.2 The platelet concentration in PRP can be up to 16 times that of whole blood [5], and it contains high concentrations of growth factors, mainly platelet-derived growth factor (PDGF), transforming growth factor (TGF)?β, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF). The concentrations of PDGF, TGF?β, VEGF and EGF in PRP have been confirmed by enzyme-linked immunosorbent assay to be three to eight times higher than the normal concentration in vivo [6]. In addition, the fibrous network within PRP has a role in promoting cell adhesion and preventing cell loss.
1.1.3 Currently, PRP-based growth factors have been successfully used in clinical applications, but they are all patient’s own PRP. if the patient isolates too much blood, then it can lead to anemia or affect the health. To overcome this limitation, HUANG Qian [7] designed to isolate growth factors from allogeneic PRP. The results showed that the growth factors isolated from PRP had four parts: A, B, C, and D. Most of them were in parts B and C, and they could effectively promote cell proliferation; the isolated growth factors, which were stored after vacuum freeze-drying, could still maintain their original biological activity.
1.2 Preparation and activation of platelet-rich plasma gel
Regardless of the principle of isolated PRP, clinically PRP is mostly applied in the form of gel, which is a viscous gel-like clot made of PRP mixed with calcium chloride and thrombin. The best proven PRP activator is a mixture of 10 mg/ml calcium chloride solution (a citrate inhibitor capable of clotting plasma) and 100 U/mL bovine thrombin (an activator that causes fibrin to aggregate into an insoluble gel, induces platelet degranulation and releases mediators and cytokines). Before use, PRP is mixed with activator in a 1:1 ratio and a little air, shaken well, and the PRP gel is made after 6-10S and spread evenly on the trauma surface, covered with biomaterial or gauze to increase the contact time of the gel with the trauma surface.
Recently, I. Martineau et al [8] showed that calcium chloride and thrombin regulate the release, synthesis and degradation of growth factors in PRP and that each growth factor has its own specific pattern. The effect of different concentrations of calcium chloride and thrombin on growth factors in PRP is very different, for example, when the concentration of thrombin is 142.8 U/ml, the concentration of EGF is at the highest level, and when the concentration of calcium chloride is 14.3 mg/ml, the concentration of IL-1 is at the highest level.
1.3 Mechanism of action of platelet-rich plasma
1.3.1 The action of PRP depends on the release of high concentrations of various types of growth factors and fibrinogen formed by the fibrous meshwork scaffold [9], including platelet-derived growth factor (PDGF), transfer growth factor-β1β2 (TGFβ1β2), vascular endothelial growth factor (vEGF), platelet-derived endothelial cell growth factor, interleukin?1 (IL?1), epidermal growth factor (EGF), fibroblast growth factor, and platelet-activating factor [10]. These factors are essential for the induction of tissue growth, and the fibrous meshwork scaffold formed by fibrinogen supports the growth factor-induced generation of new tissue. These factors play an essential role in stimulating the proliferation of osteoblasts and preosteoblasts, inhibiting osteoclast formation and bone resorption, increasing collagen synthesis capacity, promoting endothelial cell proliferation, inducing neovascularization, stimulating the division and proliferation of many types of tissue cells in vivo, promoting matrix synthesis and deposition, and promoting fibrous tissue production.
1.3.2 PRP contains high concentrations of activated growth factors to accelerate wound healing, which enables optimal synergistic effects between growth factors due to the ratio of each growth factor being similar to the normal physiological concentration in the body [11]. Meanwhile, PPP contains a large amount of fibrin, which can provide a good scaffold for repair cells, stimulate soft tissue regeneration, promote early wound closure and prevent infection [12]. Some foreign scholars [13] found that PRP also plays an important role in reducing the occurrence of inflammation through experiments on the measurement of fissure pressure after colonic suturing, and the latest study confirmed [14] that the mechanism of this effect is the release of IL?1 specific primitive inhibitory factor by macrophages in PRP to control the occurrence of early inflammation.
1.3.3 PRP gel prevents platelet loss and allows platelets to secrete growth factors locally for a long time, and these exogenous growth factors promote repair by (1) acting as chemotactic agents to chemotacticize inflammatory cells and tissue repair cells, creating conditions for wound sterilization as well as later repair; (2) acting directly on the growth factor receptors on tissue repair cells to accelerate the cell cycle transition through their pro-divisional effect to promote (3) activation of upregulated growth factor receptor activity on tissue repair cells to accelerate signaling [15].
In addition, PRP not only contains high concentrations of platelets but is also rich in coagulation factors, which under normal conditions are maintained at normal physiological levels. Activated platelets contain a large number of proteins that promote the healing process, and platelets activate these proteins within 10 minutes after coagulation, thus shortening the clinical process of healing.
2. The role of platelet-rich plasma in the healing of chronic refractory wounds
Trauma healing is a specific host immune response to intact tissue regeneration, and it has been experimentally demonstrated that growth factor activity is altered in trauma, i.e., synthesis is reduced and degradation and inactivation is increased [16]. With the topical use of PRP, traumatic bleeding sites can acquire a mixture of platelets and cold precipitates that increase exogenous growth factors.
2.1 The current study confirms that PDGF acts by increasing mobile cell proliferation as well as increasing cellular matrix products contributing to rapid granulation tissue formation. PDGF mRNA is expressed in fibroblasts and keratinocytes of the wounded surface, thus PDGF increases infiltration of trabecular fibroblasts and inflammatory cells, induces fibroblast to myofibroblast transformation in the late stages of injury, increases collagen synthesis in wound tissue, and promotes granulation tissue growth.
There are three isoforms of human TGF?β, namely TGF?β1?3, and each isoform has different spatial and post-injury temporal distribution in vivo. It has been shown that in the pre-inflammatory phase after tissue trauma, locally relevant cytokines are increased, while TGF?β levels are temporarily decreased and collagen deposition is reduced. Activated platelets produce TGF?β, which has a stimulatory effect on cell differentiation, proliferation, and inflammatory processes, and a unique role in extracellular matrix synthesis and remodeling. TGF?β reduces enzymatic degradation in wounds not only by promoting fibroblast chemotaxis, which produces collagen fibers and extracellular matrix, but also by reducing collagenase synthesis and increasing the production of metalloproteinase inhibitors, thereby promote wound healing. Experimentally, the injection of exogenous TGF?β into the wound model can activate fibroblasts, stimulate collagen fiber production, and promote wound healing, and TGF?β1 and TGF?β2 can also increase wound healing tension [17].
EGF exerts activity upon binding to receptors, which are expressed in almost all cells, but are most abundant in epidermal cells. EGF is released after topical application of PRP following skin injury. eGF not only accelerates epidermal growth, but also has the effect of increasing matrix formation and connective tissue contraction. Topical EGF has been shown to accelerate epidermal growth and increase wound healing tension in animal excision wound models. Studies have shown that both recombinant human basic fibroblast growth factor (rhbF-GF) and recombinant human epidermal growth factor (rhEGF) have trauma repair effects, comparing the two rhbFGF promotes granulation tissue production in the early and middle stages of repair, and rhEGF accelerates epithelialization of wounds in the middle and late stages [18].
IGF includes IGF?1 and IGF?2, of which the role of IGF?1 in wound repair is more studied. high concentrations of IGF?1 in PRP, when released, is a tropism of vascular endothelial cells, which can stimulate vascular endothelial cells to migrate to the site of trauma and promote neovascularization. IGF?1 can also promote the growth of many cells, such as fibroblasts, osteocytes and chondrocytes. In addition, IGF?1 can act synergistically with PDGF to increase epidermal and endothelial regeneration.
VEGFs include VEGFA, B, C, D, E and placental growth factor. VEGFA, which was first identified, has the effect of promoting vascular neovascularization and enhancing vascular permeability. vEGFA binds to endothelial cell surface receptors VEGFR1 and VEGFR2 to promote NO synthesis by endothelial cells and activate vascular neovascularization. vegfa also promotes endothelial cell division. vegfc binds mainly to VEGFR3 receptors on lymphatic vessel endothelial cells, but also to VEGFC binds mainly to VEGFR3 receptors on lymphatic endothelial cells and also to VEG?FR2. VEGFD is similar to VEGFC in that it promotes vascular and lymphatic vessel neogenesis.
2.2 In experimental and clinical studies, PRP has shown good effects in promoting the repair of difficult-to-heal wounds. in the experiments of repairing equine calf wounds with PRP, Carter found that the repair effect of PRP was significantly better than that of the control group, and concluded that the large amount of growth factors in PRP compensated for the low amount of growth factors in equine calf wounds, which started the repair mechanism faster, formed epithelial tissue, accelerated vascular regeneration, and provided a better environment for wound repair. The PRP provided a better environment and blood supply for the repair of the wound. Also, in the experiment, it was found that the PRP-treated group had less wound scarring, probably due to the high content of leukocytes and monocytes in PRP, which inhibited the inflammatory response at the wound, resulting in less scarring [19].
However, Monteiro [20], in an experiment using PRP to repair wounds on the distal forelimbs of horses, found that PRP was less effective in small wounds and more suitable for the treatment of large tissue defects by wound histology, measurement of TGFβ1, assessment of biological material, and detection of collagen I and III mRNA, and was also considered to provide a new avenue for the treatment of chronic wounds. Crovetti [16] treated skin ulcers with PRP and found that PRP better formed granulation tissue at the wound site and promoted complete regeneration of wound epithelial tissue compared to the control group. the local release of multiple growth factors by PRP together promoted wound recovery, such as the chemotactic effect of TGF?β on neutrophils and monocytes mediating the inflammatory response of the wound, PDGF stimulating fibroblast fibroblast proliferation and differentiation to promote tissue remodeling, and VEGF to accelerate vascular regeneration, but it has not been confirmed whether patients in the treatment group had significantly less pain compared to the control group.
Lee [21] et al. studied and evaluated the promoting effect of PRP in the treatment of total skin defects in rabbits. Three treatment doses of 0.3 ml,0.6 ml,0.9 ml were divided into three groups, and the rate of wound epithelial formation, wound shrinkage, tissue filling and volume fraction of fibroblasts were measured after one week and two weeks, and the results showed that PRP heals the total skin by accelerating epithelial migration and vasogenic response and reducing the wound area; it was also suggested that the treatment interval of PRP could be changed by and treatment dose to explore and evaluate the long-term effects of PRP when treating wounds.Hom [22] et al. conducted a prospective study using PRP for the treatment of full dermal defect wounds and showed that the wound healing was faster and scar growth was less pronounced in the group using PRP compared to the group using antibiotic ointment and the group using semi-exposure therapy.
GUO et al [23] performed a clinical observation on 47 chronic refractory wounds of the lower extremities (all unhealed after 2 to 4 months of treatment): the wounds were cleared and injected with autologous PRP gel once or twice every two months with a follow-up of 4 months, which showed a significant improvement in soft tissue circulation and massive granulation tissue growth in the wounds after 2 months; the wound healing rate was higher than 79.3% and statistically significant after 4 months The wound healing rate was higher than 79.3% and statistically significant after 4 months. Marquez et al [24] applied autologous PRP to 10 patients with chemical burns of the eye, with subconjunctival injection of PRP in the treatment group and conventional method in the control group. In addition, it can strengthen the cell proliferation and differentiation, and promote the healing of wounds.
3. Advantages and problems in the development of platelet-rich plasma
3.1 The current clinical use of PRP, a platelet concentrate obtained by centrifuging autologous whole blood, has received much scholarly attention in recent years. Studies have confirmed that PRP has many unique advantages: first, PRP is autologous, which fundamentally solves and avoids the concerns of immune rejection, disease transmission and possible alteration of human genetic structure by exogenous growth factors. Secondly, PRP is easy and fast to prepare, the method is more mature, the detection means are standardized, and the quality of preparation is guaranteed. Third, PRP contains a variety of high concentrations of growth factors, and the ratio of each growth factor is similar to the normal ratio in the body and has the best synergistic effect, which to a certain extent makes up for the shortcomings of single growth factor therapy. Fourthly, PRP can be coagulated into gelatinous form by thrombin, and the gelatinous PRP can not only bond the tissue defect, but also prevent the loss of platelets, so that platelets can secrete growth factors locally for a long time and maintain a high concentration of growth factors. Fifth, PRP preparation is less damaging to the patient, and only blood needs to be taken from the patient’s vein. Foreign countries have now produced a special PRP production machine, can first take blood from the vein, after extracting high concentration of platelets will be the remaining blood components back into the body, this method is low cost, can reduce medical costs. Sixth, so far, no adverse effects of PRP on the organism have been found.
3.2 The disadvantages of PRP, which greatly limit its clinical application, are.
(1) Preparation in an open system and transfer in multiple containers are vulnerable to external contamination;
(2) Platelets are susceptible to destruction or activation by exogenous stimuli in vitro, such as prolonged blood draws, small needles, application of tourniquets, inappropriate anticoagulants and blood depositors, degree of shaking, and placement time, all of which can artificially cause platelet activation;
(3) The relationship between platelets and growth factor concentration is complex and may be influenced by a variety of factors during the production process, and may also vary depending on the mode of platelet activation (freeze-activated, thrombin-activated), the degree of activation, and the sensitivity of the growth factor assay kit;
(4) The lifespan of platelets and their growth factors in vivo does not exceed 5 days, and even if PRP is activated by thrombin or calcium ions to form a gel block, the continuous release of platelet contents (including growth factors) in the gel block is only about 1 week, and it is impossible to act on the whole process of tissue repair.
3.3 Of course, there are many issues that need to be addressed in the future, such as.
(1) To further establish efficient and stable PRP production methods, to study the biological properties of growth factors secreted by PRP made by different methods and with different concentrations of activators, to clarify the growth factors secreted by PRP gel in vivo and the interactions between various growth factors, and to determine the optimal therapeutic concentration and treatment time of PRP for wound repair;
(2) Isolation of growth factors from allogeneic PRP has received wide attention, and theoretically this method can reduce immunogenicity and prevent immune rejection, but further research is needed to confirm this [7]; (30 Research on different PRP products to adapt to different clinical use conditions and to specify the production technology process and product safety issues [25]. It is believed that along with the continuous research on PRP, the application of PRP will be more widespread and convenient.