Key Words: Platelet Rich Plasma, Wound healing cascade, Growth factor, Leukocyte, Osteoarthritis, Tendinopathy, Physiology, Hemostasis, Inflammation, Proliferation, Remodeling, Regenerative medicine.
PRP is a multi-cellular platelet concentrate derived from autologous whole blood, with platelet concentrations elevated above baseline whole blood levels[1,2]. Platelets within PRP contain a high concentration of bioactive factors that are stored inside of granules that can be released to activate acute inflammation and stimulate the healing cascade[3-7]. Depending upon how PRP is generated it can contain additional blood cell types such as white blood cells (WBCs), red blood cells (RBCs), and circulating stem cells, which can all contribute to the bioactivity and healing potential of the PRP[8,9]. Plasma is the straw colored fluid component of whole blood that suspends the cellular components and contains clotting factors and essential nutrients.
KEY CONCEPT: Platelets are not the only active component in PRP. All components of whole blood have important roles in the physiology of wound healing
1.Transport of nutrients, metabolic waste products and endocrine factors.
2.Transport of blood cells.
3.Provide clotting factors such as fibrinogen and pro-thrombin to support coagulation
Platelets: Platelets are non-nucleated cells derived from very large precursor cells called megakaryocytes that reside in the bone marrow. During their development, platelets obtain large numbers of storage granules that contain different growth factors, cytokines, and hormones required for wound healing. [4,5,7,26,27] Platelet activation is a highly regulated process that culminates in degranulation, or the release of granule contents.[5,28-32] The process of degranulation is a key step in wound healing because the growth factors and other mediators that platelets release program damaged tissue for repair.[29- 34] In a normal healthy state, platelets and WBCs circulate in inactive forms. However, in pathological states, such as an injury involving blood vessels, platelets become activated by contact with components of the extravascular connective tissues that are exposed at the site of injury.[35,36] Collagen and Tissue Factor are examples of tissue components that activate platelets. 
KEY CONCEPT: Platelets and leukocytes have coordinated and cooperative activities in normal wound healing that limit acute inflammation and trigger tissue repair.
Under injury conditions, platelets have two essential functions for survival.
1.Hemostasis: Drive clotting to stop the bleeding. [17,38]
2.Inflammation: Initiate healing by releasing growth factors and bioactive molecules that activate acute inflammation and program tissue repair. [11,17,39-41]
WBCs and platelets become activated together in a physiological context for wound repair. [6,17,42] This coordinate activation facilitates crosstalk that permits the modulation and control of each other’s activities. This results in a balanced and biologically optimized tissue repair response to injury. [6,17,39,42]
WBCs: The primary role of WBCs is to decontaminate the wound and prevent infection. WBCs also debride the wound of dead and damaged tissue, and ultimately deposit and activate growth factors that direct the conversion of a fibrin clot into vascularized and viable tissue. [30,40,41,43-47] There are two main classes of WBCs.
1.Granulocytes: Include neutrophils that engulph foreign bodies, produce immune regulating cytokines, lipids, and proteases, and release antimicrobial granules. [44,48-51]
RBCs: The primary role of RBCs is to carry oxygen to tissues to support metabolism, and carry carbon dioxide waste away to prevent acidification. In wound healing, RBCs act in an amplification loop to increase the activation and release of bioactive factors from platelets. [15,22,23,52]
Peripheral Blood Stem Cells: Multipotent mesenchymal stem cells (MSCs) are capable of differentiating into multiple cell types. MSCs also secrete factors that control inflammation and promote tissue repair. [53-59] These MSCs co-localize with leukocytes based on their density into the buffycoat fraction of centrifuged blood. Therefore, leukocyte poor PRP products are also lacking in peripheral blood stem cells.
1.Hemostasis- clot formation.
2.Acute Inflammation- Platelet activation and immune mobilization 3.Proliferation- Cell multiplication and matrix deposition.
4.Remodeling- Scar formation and tissue restoration.
Each phase is dominated by a particular cell type that prepares the tissue for the events in the next phase (Figure 3). [30,40,45- 47] It is important that each phase is executed effectively to ensure transitions between phases occur properly. If this does not happen, the repair process may be subverted into chronic and potentially degenerative pathological states.[39,41,46,49,61-63]
KEY CONCEPT: Thrombin represents a critical hinge between hemostasis and acute inflammation. RBCs play an important role in amplifying thrombin generation to ensure efficient and effective execution of both phases of the wound healing cascade.
[38,70] Partial activation of platelets by collagen causes a shape change that makes platelets stick together (aggregation) and adhere to the site of injury.[37,46,70,71] Aggregation stimulates platelets to begin releasing factors that reinforce their aggregation and adhesion, and promote further recruitment of platelets into the forming clot known as a “platelet plug” or thrombus.[35,72] Among the platelet factors released during the initial hemostasis response, ADP (adenosine di-phosphate) and LPA (lyso-phosphatidic acid) are particularly important becausethey can activate RBCs. [22,23,52,73,74].
Activated RBCs influence three important actions that contribute critically to the healing cascade.
The evolutionary pairing of hemostasis and acute inflammation occurs through the action of thrombin. Thrombin helps to ensure the acute inflammatory response is proportional to the magnitude of the injury.[81,82] Maintaining balanced wound healing physiology is an important concept as insufficient inflammation may result in poor or delayed healing, while excessive inflammation may interfere with healing by causing additional tissue damage. [45,46,62]
The acute inflammatory phase of the healing cascade begins in earnest when platelets become fully activated by thrombin and degranulate, releasing inflammatory mediators and growth factors from platelet storage granules.[4,5,30,83,84] Platelets contain three types of storage granules. [5,83]
a.Lysosomes: Contain digestive enzymes
b.Dense Granules: Contain small signaling molecules like Calcium ions, ADP and ATP.
c.Alpha granules: Contain hemostatic factors, growth factors and inflammatory mediators.
Alpha granules are the largest and most prevalent granule type in the platelet. These granules serve as storage vessels for effectors of the healing cascade. Platelet growth factors released from alpha granules are largely responsible for the role that platelets play in directing injury repair and wound healing (Table 1).[4,5,7,24,30,72,83,84] Platelets release their alpha granule contents with an initial burst, and exhaust most of their stored cargoes within the first hour after activation.
platelet growth factor
Stimulates cell proliferation
Potent fibroblast and immune cell recruitment factor
Promotes extracellular matrix synthesis
Potent immune suppressor
Stimulates endothelial cell proliferation
Promotes cell differentiation and maturation
Promotes collagenase activity and tissue remodeling
SDF-1α / CXCL12
Potent stem cell recruitment factor
Monocyte recruitment factor
Neural cell progenitor recruitment factor
Augments angiogenic effect of VEGF
KEY CONCEPT: Activated platelets and primed neutrophils cooperate in the biosynthesis of factors that control the magnitude and duration of inflammation.
An additional cooperative lipid-mediated antiinflammatory pathway between activated platelets and platelet primed neutrophils has also been identified (Figure 5).[79,89,90] Thistrans-cellular metabolic pathway involves the generation of arachidonic acid derived lipid mediators that modulate inflammation and promote wound healing.[79,89,90] More specifically, the attachment of activated platelets to neutrophils allows neutrophils to take up arachidonic acid that is released by activated platelets. Neutrophils can convert this platelet-derived lipid to various prostaglandins (PGs) and leukotrienes (LTs). PGs can contribute to pain and tissue swelling, while LTs typically act as potent chemotactic signals for the recruitment of immune cells.[92,93] However, platelets attached to neutrophils that generate LTs can quickly take up these inflammatory mediator lipids and convert them to lipoxins (LXs). Lipoxins are very potent anti-inflammatory molecules that play important roles in limiting neutrophil activation, preventing their migration from vessels into tissues, and in driving the resolution of inflammation. [79,89,90,94] It is important to note that platelets lack the ability to synthesize lipoxins without the LTs intermediates produced by neutrophils. In addition, neutrophils can be induced by PGs to switch the arachidonic acid lipid metabolites they generate from pro-inflammatory LTs directly to LXs.[95,96] This switch from generation of pro-inflammatory lipid mediators to antiinflammatory lipid mediators in neutrophils is thought to prevent further neutrophil recruitment and inflammatory activation while simultaneously activating resolution pathways that can accelerate healing. [79,90,91,96] Transcellular cooperative regulation of the magnitude and duration of the inflammatory response may begin to explain why platelet-leukocyte ratios are emerging as important parameters in the therapeutic efficacy of platelet-rich plasma products.[34,44,66,76,97,98]
KEY CONCEPT: Neutrophils require separate priming and activation signals to elicit an inflammatory response. In the context of an aseptic tissue injection there is no separate neutrophil activation signal available.
In the case of leukocyte-rich PRP without prematurely activated platelets, WBCs have been harvested from the circulation where they are in a resting state. Therefore the WBCs in the PRP remain in a resting state when they are introduced to the tissue. In this case, WBC priming occurs upon collagenmediated platelet activation within the treated tissue.[11,17,83] Therefore, all aspects of the pathological state within the tissue being treated are simultaneously part of the leukocyte priming reaction. Thus, no separate activating signal is present to drive the injected WBCs to an excessive inflammatory state. This theory sheds light on why autologous platelet rich plasma preparations do not present a significant safety risk when used therapeutically, regardless of their leukocyte content.[1,31-34,44,112-116]
In the primed but not activated state, WBCs have enhanced phagocytic activity that promotes debridement of damaged tissue and the release of a large array of immunomodulatory and anti-inflammatory mediators such as TGF-b1 and IL-1RA as well as lipid modulators of inflammation like lipoxins and resolvins.[79,90,117]
In contrast, neutrophils contained in a PRPinjection, for example, are in a resting state when introduced to the tissue and still require priming before they can be activated to launch an inflammatory response. Priming can be accomplished when platelet activation and degranulation occurs within the injected tissue. In this scenario, platelet-driven neutrophil priming would occur in the context of whatever inflammatory mediators are also present in the injected tissue. Therefore, no additional neutrophil activating signals are present and so the injected cells remain in the primed, but not activated, state. In this state, neutrophils have an enhanced phagocytic activity and are competent to cooperate with platelets in controlling the magnitude and duration of inflammation by producing STOP signals that prevent further immune cell recruitment from the circulation and the surrounding tissues. This activity promotes resolution of the inflammatory phase and accelerates the transition to the proliferation phase of wound healing.
The requirement for separate WBC priming and activation signals and the anti-inflammatory activities of primed but not activated neutrophils can in part explain the clinical literature regarding the emerging greater efficacy of leukocyte rich PRP treatment compared with leukocyte poor PRP to relieve pain and improve function in osteoarthritis and in tendinopathy patients.[113,114] Indeed, patients with an array of soft tissue pathologies have been shown to benefit from LR-PRP injections without any significant associated adverse events despite containing concentrated leukocytes.[113-115]
KEY CONCEPT: Protease activity does not simply destroy extracellular matrix and cause degeneration. Protease cleavage inactivates pain mediators and inflammatory cytokines, and activates growth factors for proliferation, angiogenesis, and new matrix synthesis.
Protease activity from macrophages and fibroblasts also becomes critical at this point in the healing cascade as the fibrin matrix must be partially digested to allow the migration of cells in the wound.[45,46,123] Additionally, key growth factors such as IGF-1, TGF-b1 and VEGF depend on extracellular protease activity for their bioavailability.[123-125] Moreover, protease cleavage also targets the destruction of key inflammatory mediators like IL-1b and MCP-3 (CCL7), and as such functions to dampen inflammation and promote transition to the proliferation phase. [50,118,125] Therefore, non-selective strategies such as α2- macroglobulin therapy that aim to inhibit protease activity to improve the strength of the matrix may in advertently interfere with tissue repair and delay healing. [123,125] Interestingly, of the matrix metalloproteinases, MMP-2 (neutrophil gelatinase A) activity in particular appears to play key functional roles in promoting the growth of new blood vessels and in driving wound resolution. This may be in part due to the ability of MMP-2 to inactivate inflammatory mediators like IL-1b and MCP-3 and to release critical growth factors like TGF-b1 and IGF- 1 from their tissue inhibitors.[48,106,123-127] The catalytic activity of MMP-2 also unlocks the anti-inflammatory potential of TGF-b1. [48,106,124,126] In addition, MMP-2 can also inactivate powerful neuro-inflammatory mediators of chronic pain such as Calcitonin Gene-Related Peptide (CGRP). CGRP is thought to play an important role in the chronic pain of osteoarthritis by causing vasodilation that contributes to effusion, and by causing persistent sub-acute synovitis and hyperalgesia.[129,128] It is therefore noteworthy that recent data indicate that levels of active MMP-2 are greater in leukocyte-rich PRP releasates compared with leukocyte poor. This finding should not be considered surprising since neutrophils represent the richest source of MMP-2, also known as neutrophil gelatinase A.
Granulation tissue consists primarily of fibroblasts and new blood vessels.[39,40,45-47] Two fundamental processes, provisional matrix deposition and the formation of new blood vessels (angiogenesis), work in parallel to drive repair during the proliferation phase. Angiogenesis lags slightly behind new matrix formation.[40,45-47,131] Oxygen is critical for fibroblasts to produce collagen in order to establish granulation tissue. Thus, re-vascularization is a key rate-limiting step in the healing cascade. A wide range of growth factors and chemical mediators have been identified that influence the developing capillaries. These include macrophage derived factors like TGF-b1, PDGF, VEGF and FGF.[39,40,44-47,131,132]
In the wound, fibroblasts initially produce type III collagen, which provides a weaker and less extensively cross linked tissue matrix than the type I collagen of the uninjured or mature repair tissue. Collagen III stabilizes the forming granulation tissue, but it is easier for fibroblasts, endothelial and immune cells to migrate through. This facilitates repopulation of the granulation tissue with viable cells. Type III collagen will be replaced in the matrix with type I collagen as healing progresses from proliferation to remodeling. [39,40,46,47,62,131]
Towards the end of the proliferation phase, fibroblasts are driven by macrophage signals, predominantly TGF-b1, to transdifferentiate into myofibroblasts.[49,133-135] Myofibroblasts are specialized cells that generate new matrix but also become contractile through the expression of smooth muscle actin. Contraction is important because it provides mechanical strength to the granulation tissue and reduces the wound size.[133-135] An interesting property of myofibroblasts is that contraction triggers their generation of TGF-b1. [136,137] This autocrine stimulation reinforces the myofibroblast phenotype and drives other fibroblasts to transdifferentiate too. [135,136] This autocrine TGF-b1 signaling also augments collagen I production from the myofibroblast. The new collagen I fibers are deposited in bundles that align with the direction of myofibroblast contractile force, thus strengthening the tissue and reinforcing it to resist mechanical shear stress.[133-136] At this stage of wound healing, myofibroblasts begin to degrade the provisional collagen III matrix primarily through the action of matrix metalloproteinases, and this marks the transition from proliferation to the remodeling phase where the granulation tissue will mature into a scar. [123,125,133]
1.Replacement of type 3 collagen with type 1 collagen.
2.Re-alignment of type 1 collagen fibers along the myofibroblast axis.
3.Resorption of excess neuro-vascular networks and myofibroblasts restores the tissue to function.
Remodeling is the last phase of wound healing and occurs from day 21 up to one year or longer after injury.[39,40,45-47] During the remodeling phase, formation of new granulation tissue ceases as fibroblasts either die through apoptosis or differentiate into myofibroblasts. The failure to properly transition from the proliferation phase may lead to excessive or hypertrophic scarring. [46,49,123,131] Resorption of the vascular capillary network and its associated neural network are hallmarks of a mature scar tissue. Thus, the completely remodeled and fully mature scar tissue is relatively acellular and avascular.
The remodeling phase primarily involves the refinement of collagen and its associated extracellular matrix. At this stage of healing collagen synthesis and destruction both occur at a greater rate compared with normal tissue.[133,134,136,138] Under normal physiological conditions, the maturing scar is a very dynamic system where the balance of anabolic and catabolic processes ultimately favors the maturation of the scar into a functional connective tissue. The normal outcome of the wound healing cascade is a functional tissue, a mature scar will have around 80% of the strength of the original tissue.[46,49,133,138]
KEY CONCEPT: None of the components of whole blood functions alone in the normal physiology of wound healing. Cellular co-operativity is important for execution at each phase of the healing cascade.
Platelet rich plasma can be prepared using various methods that will either maintain physiological ratios of blood components (leukocyte rich PRP containing RBCs), or remove specific components such as RBCs and WBCs (leukocyte poor PRP), or even just RBCs (Pure PRP). A considerable amount is known about how blood components contribute to normal wound healing however, very little is actually known about how PRP may influence this cascade of signaling events. Historically, platelets have been considered as the active component in PRP, as these are the most abundant of the blood cells that contain storage granules for growth factors and immunomodulators. However, it has been demonstrated that platelet function is compromised when other blood components are missing from PRP. Therefore, it seems logical to conclude that the complete physiological repertoire of blood components, rather than any individual part, should be considered the true “active component” of PRP. Until clinical studies advance to the level of sophistication that allows for the elucidation of an “optimal formulation” of PRP, maintaining the physiological context of platelets with respect to other blood cells may be one way to ensure robust and balanced PRP activity. This may indeed represent the optimal means for harvesting the efficient wound healing potential of blood, which has evolved over many millennia.
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