Platelets and Platelet-Inspired Biomaterials Technologies in Wound

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Platelets and Platelet-inspired Biomaterials Technologies in Wound-healing Applications Ujjal Didar Singh Sekhon, and Anirban Sen Gupta ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 24 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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ACS Biomaterials Science & Engineering

Platelets and Platelet-inspired Biomaterials Technologies in Wound-healing Applications Ujjal Didar Singh Sekhon1, Anirban Sen Gupta1* 1

Case Western Reserve University, Department of Biomedical Engineering, Cleveland, OH 44102, USA

* Corresponding author: Dr. Anirban Sen Gupta Case Western Reserve University Department of Biomedical Engineering 10900 Euclid Avenue Wickenden Bldg, Rm 517B Cleveland, OH 44106 Phone: 216-368-4564 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract Wound healing is a complex biological process involving distinct phases of hemostasis, immune response and inflammatory events, regulated cellular proliferation and matrix remodeling. While immune and inflammatory cellular phenotypes (e.g. neutrophils and monocyte/macrophages) are often the focus of wound healing studies, the initial hemostatic and sustained secretory role of platelets to modulate the various mechanistic phases of wound healing via clot promotion, clot stabilization and retraction, release of various growth factors and cytokines from active platelet granules, and release of matrix remodeling enzymes, are becoming exceedingly appreciated in pre-clinical and clinical settings. This has led to extensive studies using platelet-based products like platelet-rich-plasma (PRP) suspensions and gels as topical and injectable technologies to augment wound healing in both soft and hard tissues. In parallel, a robust volume of research is currently being directed at mimicking and leveraging the hemostatic and secretory mechanisms of platelets utilizing various lipidic and polymeric biomaterials systems. The current article is aimed at providing a review of platelet’s involvement in wound healing mechanisms and subsequently discuss the current stateof-art regarding various platelet-based as well as biomaterials-based approaches and technologies to promote wound healing.

Keywords: Platelets, Wound healing, Growth factors, Cytokines, Biomaterials, Drug Delivery


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A. Introduction Wound healing and tissue regeneration encompass a complex concert of coagulatory, inflammatory and immune mechanisms for restoration of normal physiological function through repair and regeneration of human cells, tissues or organs1. In mammals, wound healing occurs in four unique yet overlapping stages of (i) coagulation/hemostasis (ii) inflammation/migration (iii) proliferation/angiogenesis/regeneration and (iv) tissue remodeling2. Current approaches to facilitate these stages of wound healing involve the use of various biomaterials-based matrices, growth factors and cell-based therapies (stem cells or differentiated cells) to treat a variety of wounds and tissue insults including truncal and limb tissue injuries, spinal cord and brain injuries, heart muscle damage, Parkinson’s disease, Alzheimer’s disease etc.1,3,4. Interestingly, several of the current molecular mechanistic components of wound healing and regenerative treatments can be traced back, at least in part, to be derived from platelets5. Platelets are anucleated blood cells with a circulation life-time of 7-10 days and primarily contribute to physiological hemostasis and pathological thrombo-inflammation6,7. ‘Resting’ platelets circulating in the blood stream become activated when they come in contact with sub-endothelial matrix proteins and tissue factor-bearing cells exposed upon tissue injury and vascular disruption. Through a combined effect of adhesion and aggregation mechanisms, they begin to form a platelet plug that ultimately mediates and amplifies the coagulation cascade mechanisms to form the fibrin mesh characteristic of a blood clot

6,8–11

. Recent research has revealed the mechanistic contribution of

platelets in various physiological aspects of signal transduction, inflammation, healing and tissue regeneration in response to injury12–14. Platelets’ primary involvement in



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hemostasis and coagulation provides them with the ideal location to subsequently contribute to various spatio-temporal aspects of wound healing. For example, platelets can directly amplify the generation and stabilization of the fibrin matrix that forms the bulk of the wound bed in the initial stages of hemostasis and inflammatory activities. Within the hemostatic plug, activated platelets release dense granules and α-granules which contain a variety of growth factors, chemokines, cytokines and pro-adhesive molecules that can promote inflammatory and regenerative cell recruitment, proliferation and differentiation15. Platelet granule contents can also help regulate angiogenesis via release of pro-angiogenic and anti-angiogenic factors16. The abundance of growth factors in the platelet secretome has resulted in platelets becoming a unique treatment source in surgical and clinical applications that require tissue regeneration15. The following sections will describe platelets and their secretome, review the mechanistic involvement of platelets in wound healing and tissue regeneration, and provide critical insights on natural platelet-based and potential synthetic platelet-based biomaterials technologies to promote wound healing.

B. Platelets: Surface Molecules and Secretome Components Platelets are essentially the ‘first cellular responders’ to the injury/wound site, and they linger on through much of the inflammatory phase. This gives them the ability to act as a unique platform to spatio-temporally influence multiple aspects of wound healing. In order to appreciate a platelet’s short and long term role in the wound healing mechanism, it is important to describe a platelet’s surface components, as well as, cytoplasmic and granular compositions.


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B.1. Platelet Surface Molecules Platelets are anucleated blood cells shed from mature megakaryocytes in the bone marrow into blood circulation, by a process historically termed ‘thrombopoiesis’17,18. Human platelet count is normally in the range of 150,000 - 450,000 per microliter of circulating blood. Circulating platelets are of bi-convex discoid morphology, about 2-3 µm in diameter and 0.5 µm in thickness19. The surface of platelets is rich in a variety of receptor proteins that play crucial roles in a platelet’s mechanistic involvement in hemostasis20,21. Specifically, platelet surface receptor component GPIbα of the GPIb-IXV complex mediates binding to von Willebrand Factor (vWF), and surface receptors GPIa-IIa (also known as integrin α2β1) and GPVI mediate binding to collagen. A combination of these mechanisms allows platelets to undergo adhesion at a bleeding wound site. Platelet surface also contains receptors for multiple agonists, e.g. P2X1 for ATP, P2Y1 and P2Y12 for ADP, PAR-1 and PAR-4 for thrombin, and TXA2R for thromboxane. Outside-in signaling induced by platelet adhesion to vWF and collagen, along with agonist action on platelets, result in platelet activation where platelets transform from ‘discoid’ morphology to ‘pseudopodal stellate’ morphology. The platelet surface also bears a high density of receptor GPIIb-IIIa (also known as integrin αIIbβ3) which assumes a ligand-binding conformation upon platelet activation, and can bind to specific domains of blood protein Fibrinogen (Fg) as well as locally deposited vWF. The Fg-to-GPIIbIIIa binding interaction is the primary mechanism that drives interplatelet bridging to facilitate aggregation of active platelets. These adhesion and aggregation mechanisms together promote formation of platelet plug at the bleeding injury site in hemostasis. Figure 1 shows (A) a schematic representation of important platelet surface



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molecules relevant to hemostasis along with their respective ligand/substrate components, and (B) shows representative SEM images of inactive ‘discoid’ platelet transitioning into activated ‘pseudopodal stellate’ platelet and finally into fully active ‘spread’ morphology that is characteristic of their morphological transition during hemostatic action. This morphological transition also allows platelets to undergo cell-cell and cell-matrix interactions over higher surface areas. During and after hemostasis, activated platelets release a variety of cytoplasmic granules and granule contents, lysosomal contents, microparticles and exosomes, and these further act as depots for immediate or sustained release of growth factors, adhesive proteins, cytokines and other signaling molecules that play important roles in regulating wound healing and tissue repair14,22–24.

B.2. Platelet granules and secretome components Platelets have a variety of cytoplasmic and lysosomal storage granules rich in adhesive molecules, growth factors, chemokines, cytokines and various other signaling molecules. These are collectively released as ‘platelet secretome’ upon platelet activation and modulate various aspects of hemostasis and post-hemostatic events13,25. Figure 2A shows a representative electron microscopy image of a platelet’s cytoplasmic ultrastructure, 2B shows a cartoon schematic of these ultrastructural components and 2C shows the various platelet secretome biomolecular components tabulated according to their involvement in characteristic phases of wound healing. These components have a diverse effect on the biochemical, molecular and cellular environment at the wound site, in the context of inflammation, recruitment of neutrophils, macrophages and


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fibroblasts, anti-microbial response, angiogenesis, extracellular matrix synthesis and maturation, etc. The following sections will provide distinct descriptions of characteristic platelet granules, their secreted components and other miscellaneous signaling components.

B.2.a. Platelet α-granules Platelet α-granules are the most abundant platelet cytoplasmic granules, comprising more than ten times the cytoplasmic volume as compared to dense granules. α-granule size varies from 200-500 nm and they house a variety of adhesive proteins, chemokines, cytokines and growth factors13,16,26,27. These granules are formed in megakaryocytes and later transported to platelets which, upon activation, allow the αgranules to fuse with the platelet cell membrane and trigger the release of granule contents28. Typical adhesive proteins found in platelet α-granules are fibrinogen, fibronectin, vWF and thrombospondin-1, which play an important role in the hemostatic phase13,29. Some adhesive receptors such as the integrin αIIbβ3 (receptor for Fg), GPVI (receptor for collagen) and GPIbα-IX-V (receptor complex for vWF) are also found inside α-granules, that may add to the already existent expression of receptors on the platelet surface

30,31

. Small amounts of important coagulation factors such as Factor V

(forms a part of the prothrombinase complex) and Factor XIII (crosslinks soluble fibrin upon activation) have also been reported to be released from α-granules32. These granules also contain important cell adhesion molecules (CAMs) like the membrane glycoprotein P-selectin, that can translocate to the platelet membrane upon platelet activation (and granule fusion) and are involved in platelet interactions with surrounding



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platelets, as well as, recruitment of and interaction with neutrophils, monocytes and lymphocytes16,33. In addition, these granules contain inhibitory proteins and enzymes like plasminogen activator inhibitor-1 (PAI-1), α2-antiplasmin, protein S and tissue factor pathway inhibitor (TFPI), that help regulate hemostasis by inactivating pro-coagulant factors over time as the later phases of wound healing begin32,34,35. Chemokines and cytokines released from α-granules vary from being microbicides to regulatory factors for angiogenesis. For example, CXC chemokine ligand 4 (CXCL4, also known as Platelet Factor 4) facilitates recruitment of neutrophils and fibroblasts and can also initiate the endothelial adhesion and degranulation of neutrophils, along with the activation and differentiation of monocytes into macrophages36–38. CXCL4, along with other secreted chemokines such as thymosin-b4, PBP, CTAP-III, NAP-2 and CCL5 (RANTES), all released from platelet α-granules, can defend the wound healing process against microbial infection39,40

41

. The chemokine, RANTES, also plays a role in

monocyte arrest and along with other chemokines, such as IL-8, regulates the production of inflammatory molecules by endothelial cells14,36. The CD40 ligand (CD40L), upon translocating from the α-granule membrane to the platelet membrane, acts as a substrate for matrix metalloproteases to form soluble CD40L (sCD40L) and can promote various inflammatory and immune responses (both innate and adaptive), since the receptor for CD40L is present on platelets, neutrophils, endothelial cells, Tcells, B-cells, macrophages and dendritic cells42,43. To aid the antimicrobial defense function, complements and complement precursors have also been reported to be released by α-granules to participate in the complement activation cascade44.


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α-granules also store several growth factors that directly and indirectly promote cell recruitment, transformation/maturation and proliferation in the inflammatory phase of wound healing. One of the most important growth factors released is the plateletderived growth factor (PDGF) which acts as a chemoattractant for endothelial cells and promotes cell proliferation, especially in the process of generating new blood vessels (i.e. angiogenesis) where it works in concert with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)45. Another biomolecule released from α-granules, transforming growth factor β1 (TGF-β1), is essential in wound healing and scarring where it promotes signaling in fibroblasts for ECM production, recruits inflammatory cells to the wound area and can also initiate the production of VEGF from platelet α-granules46,47. VEGF is important for the formation of new blood vessels and also acts as a pro-inflammatory factor by allowing endothelial cells to adhere leukocytes onto them. CXCL4 secreted from platelet α-granules can negatively regulate angiogenesis and inhibit the activity of growth factors such as VEGF48. Other growth factors such as hepatocyte growth factor (HGF), epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) released from α-granules are also involved in the induction of cell proliferation via activation of fibroblasts14. Among other molecules released from α-granules that have relevance to wound healing are matrix metalloproteinases (MMPs), e.g. MMP-1, MMP-2, MMP-9 and ADAM-10. These granules also contain tissue inhibitors of metalloproteases (TIMPs 1-4) and antiangiogenic proteins such as angiostatin and endostatin49,50. Recent studies suggest that pro-angiogenic and anti-angiogenic factors may be present in differently packaged subsections of α-granules and their release might be agonist-dependent49,50. These



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studies also indicate that platelets (and their secretome) therefore can positively and negatively regulate the angiogenesis processes associated with wound healing in the late inflammatory phase.

B.2.b. Platelet Dense granules Platelets have 3-8 dense granules per platelet and these granules contain Adenosine TriPhosphate (ATP), Adenosine DiPhosphate (ADP), Guanosine diphosphate (GDP), serotonin, calcium, magnesium, pyrophosphates and polyphosphates13,51. Dense granules undergo fast release from platelets owing, in part, to their positive feedback effect on a platelet’s ability to release dense granules. For example, ADP, Calcium and serotonin, secreted from these granules, act on platelets from the outside after their release initiating a positive feedback loop that ensures further recruitment of platelets to the injury site via activation and aggregation. While the details of dense granule release are not fully understood, it is known that when platelets are activated by thrombin receptor-activating peptide (TRAP), thrombin and thromboxane, and a synergistic balance between protein kinase C activity and elevation of Calcium levels is required to ensure dense granule release53. Calcium is an important regulator of the coagulation pathway and interacts with coagulation complexes like the prothrombinase complex to allow thrombin generation13,54. Serotonin mediates constriction and permeability in blood vessels. Serotonin also plays an important role in regeneration of organs such as the liver55. Dense granules pack polyphosphates that are 60-100 phosphate units long at a concentration of 130 mM56–58. These polyphosphates enhance coagulation by accelerating Factor V, Factor XI and thrombin activation58. The importance of dense


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granule secretions in wound healing is further underlined by the fact that their absence (or inability to release contents) affects the body’s ability to restore tissue integrity. For example, dense granule deficiency has been implicated in part in severe mucocutaneous and postoperative bleeding disorders (Hermansky-Pudlak syndrome), neurological abnormalities and neutrophil dysfunction (Chediak-Higashi syndrome), thrombocytopenia with absent radii (TAR) syndrome and Wiskott-Aldrich syndrome 51,59– 64

.

B.2.c. Lysosomal granules Platelet lysosomal granules can secrete acid hydrolases (cathepsins D and E, hexosaminidase,

b-galactosidase,

arylsulfatase,

b-glucuronidase

and

acid

phosphatase) as well as elastases and other matrix-degrading enzymes. Even though their function hasn’t been fully explored, it is known that they play a role in digestion of phagocytic and cytosolic components, receptor cleavage, fibrinolysis, and degradation and remodeling of extracellular matrix components and regulation of vasculature25.

B.2.d. Platelet Extracellular Vesicles: Microparticles and Exosomes Platelets undergoing activation and/or apoptosis, are known to shed membrane evagination-mediated microparticles, originally termed ‘platelet dust’, which contain several proteins and hemostasis promoting biomolecules65,66. Platelet microparticles (PMPs) comprise the majority of circulating microparticles in blood and are involved in hemostasis, cell-cell communication, stimulation of inflammatory pathways, regulation of angiogenesis and release of various wound healing relevant factors25,65,67. As with platelet membranes, platelet microparticles bear pro-coagulant anionic lipid content on



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their surface for the prothrombinase complex to form. Their importance in physiological hemostasis has been underlined by their role in diseases like Scott’s syndrome, arterial thrombosis, atherosclerosis and idiopathic thrombocytopenia68,69. Platelet microparticles contain both pro- and anti-thrombotic agents and this sets their role in coagulation as the equilibrium regulators. They have been shown to express varying membrane compositions (e.g. P-selectin and PS expression) based on the platelet agonist70. It has been suggested that these microparticles can promote angiogenesis, as demonstrated by their mediation of proliferation and tube formation of human umbilical vein endothelial cells (HUVEC)71. Platelet microparticles have also been reported to enhance the transfer of platelet antigens such as CD41, CD61, CD62, CXCR4 and PAR-1 to the surfaces of hematopoietic stem-progenitor cells72. Platelet microparticles also contain RANTES which has been suggested to play a role in augmenting adhesion and neovascularization capacities of impaired circulating angiogenic cells (CACs)73. Platelet microparticles have been reported to increase endogenous neural stem cell proliferation, neurogenesis and angiogenesis in ischemic brain74. Even though there is evidence-based data on platelet microparticle role in wound healing, some of the mechanisms surrounding differential release of contents and corresponding effects are yet to be fully explored. Activated platelets are also known to release cytoplasmic multivesicular bodies by direct exocytosis, and these particles are termed ‘exosomes’75. Exosomes are also secreted by other cells besides platelets e.g. T-cells, B-cells, mast cells, epithelial cells, endothelial cells etc., and are reported to be rich in tetraspanin family of proteins as well as various microRNAs76. In recent years, exploring exosomemediated physiological and pathological mechanisms and leveraging this knowledge for


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development of therapeutic platforms has emerged as an exciting area of research75. To this end, some studies have shown that platelet exosomes can influence wound healing components of in vitro and pre-clinical models, but detailed mechanisms and intricate pathways of platelet exosome mediated healing mechanisms are a subject of ongoing research77–79.

B.2.e. Proteins and Metabolites secreted from Platelets Platelets contain over 300 types of proteins and although platelets do not have nuclei, they have been reported to possess certain machinery for protein synthesis, for example, with the use of ‘spliceosome’, a complex that can process pre-mRNAs80. Signal-dependent splicing is a novel function possessed by platelets despite their anucleate nature81. Platelets can splice Tissue Factor pre-mRNA to mature mRNA, resulting in increased procoagulant activity. Cdc2-like kinase (Clk) mediates this splicing pathway to generate Tissue Factor in response to cellular activation82. Sphingosine-1phosphate is another active metabolite released by platelets that is involved in signaling pathways and will be discussed further in the next section. Platelets also play an important role in the complement cascade wherein their effect varies based on local wound conditions. For example, C3 localization is known to increase on activated platelet surfaces and P-selectin has been shown to act as a receptor for C3b leading to the propagation of the complement cascade83. Tissue Factor and CD40 are other platelet-relevant molecules that play important roles in hemostasis and wound healing relevant signaling pathways. Tissue Factor aids the conversion of Factor X to Factor Xa which forms a part of the prothrombinase complex, a major component of hemostasis84.



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B.2.f. Miscellaneous platelet-derived secreted molecules in signaling Platelet components such as α-granules and microparticles contain and transport surface receptors to the platelet membrane along with releasing signaling molecules that mediate recruitment and regulation of various hemostatic, inflammatory and angiogenic factors. Some important secondary messengers are released from activated platelets, including lysophosphatidic acid (LPA), phosphatidic acid (PA) and Sphingosine-1-phosphate (S1P) all of which are high affinity agonists for G-protein coupled endothelial differentiation gene (EDG) receptors85. S1P promotes chemotaxis and subsequent proliferation of endothelial cells resulting in early blood vessel formation. S1P works through a receptor dependent process and has also been shown to improve the ability of fibroblast growth factor in inducing angiogenesis86. A proinflammatory lipid, platelet-activating factor (PAF), is subject to spatially controlled production and helps localize neutrophils on the surface of thrombi87. Activated platelets, as well as endothelial cells, contain another adhesion receptor, P-selectin, which is important in regulating recruitment and signaling involving several different types of cells and molecules. P-selectin mediates leukocyte recruitment in inflammatory sites88. Soluble P-selectin (sP-selectin) can induce leukocyte-derived microparticle production and activation of leukocyte integrins. Elevated sP-selectin levels have been associated with abnormalities such as higher blood brain barrier permeability, altered social behavior with increased aggression, larger infarcts in the middle cerebral artery occlusion ischemic stroke model and increased susceptibility to atherosclerotic macrophage-rich lesion development89. Higher sP-selectin in blood caused increased concentrations of pro-coagulant microparticles and resulted in faster clotting times90.


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Platelets also carry the CD-40 ligand which plays a role in the up-regulation of adhesion molecules, pro-inflammatory chemokines and increase in Tissue Factor expression among other functions54. CD40L triggers platelet activation via outside-in signaling when it binds to integrin αIIbβ3 and effects tyrosine phosphorylation of β391. This in turn can influence platelet aggregation and coagulation cascade amplification at the wound site. It has also been demonstrated that platelet-derived alpha-chemokine platelet factor 4 (PF4) is able to affect the differentiation of monocytes into macrophages38. Another important aspect of wound healing is the activation of macrophages wherein they exist in either the classically activated M1 state or the alternatively activated M2 state (M2 has been reported to have three further subtypes namely M2a, M2b and M2c) and these perform mainly three kinds of activities – host defense, wound healing and immune regulation92,93. While the M1 phenotype is known to produce inflammatory cytokines (such as IL-1β, TNFα, IL-6, IL-8 etc) as well as growth factors (VEGF, FGF), the transition to the M2 state is essential for wound healing, especially since M2a macrophages contribute to extracellular matrix deposition and wound closure by generating precursors for collagen and fibroblast stimulating factor, and M2b and M2c marcophages work towards suppressing inflammation via release of IL-1094. Recent research has shown that platelets can induce certain aspects of macrophage polarization and re-polarization from the pro-inflammatory state to the repair phenotype, and thereby can, in effect, modulate wound healing phases95,96.



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C. Platelet Involvement in Wound Healing Phases Building on the descriptions of platelet surface molecules and secretome components provided in the previous sections, this section will focus on correlating the role of these molecules and components in the complex spatio-temporally regulated phases of wound healing, namely, hemostasis, inflammation, proliferation and remodeling97,98. Figure 3 shows representative schematic of these four phases.

C.1. Platelet role in Hemostasis phase Tissue injury and vascular disruption result in disruption of endothelial cell lining and exposure of sub-endothelial matrix proteins as well as tissue-factor bearing cells to blood components. In response, the coagulation cascade gets initiated via activation of Factor XII (intrinsic pathway) as well as interaction (and activation) of Factor VII with the exposed Tissue Factor (extrinsic pathway)98. Platelets interact with and adhere to the exposed sub-endothelial matrix components, especially to vWF (secreted and deposited from injured endothelium) and to sub-endothelial collagen, utilizing their GPIbα and GPIa-IIa/GPVI surface receptors respectively

6,99

. Platelet adhesion to these

components, as well as, production of small amounts of thrombin via the Tissue Factor pathway, results in platelet activation. Activated platelets further secrete granule contents in the surrounding and bridge with each other via Fg-to-GPIIbIIIa interactions to form aggregates that ultimately lead to the platelet plug at the bleeding site100,101. The surface of activated platelets expose a high amount of negatively charged phospholipids (e.g. phosphatidylserine or PS) that further promote co-localization and activation of various coagulation factors, leading to formation of the tenase and prothrombinase


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complexes, thereby amplifying the production of thrombin102,103. This in turn converts fibrinogen to soluble fibrin, which is further cross-linked by thrombin-activated Factor XIIIa into a biopolymeric mesh that secures the platelet plug and other blood components in place to stabilize the clot

6,104

. Activated platelets also secrete

polyphosphates (PolyP), an anionic linear inorganic polymer, which augments clot formation by activating Factor XI and Factor V and further amplifies the generation of thrombin.58 The resultant cross-linked fibrin mesh provides a suitable matrix for the subsequent phases of inflammation, healing and regeneration since the trapped platelets can release granules filled with growth factors, cytokines, chemokines and various other signaling molecules105. These secreted compounds aid the propagation of the wound healing cascade by activating and attracting neutrophils, macrophages, endothelial cells and fibroblasts106,107. Eicosanoids like leukotrienes, prostaglandins and thromboxanes (partly released from platelets) are produced from the breakdown of arachidonic acid and play important roles in stimulating and regulating subsequent events in the inflammatory phase98. Therefore, platelets play a primary role in staunching bleeding (hemostasis) at the wound site via adhesion, activation and aggregation at the site, augmentation of fibrin generation and stabilization, and subsequent secretion of cytoplasmic and granule contents to influence subsequent phases of wound healing, as depicted in Figure 4.

C.2. Platelet role in Inflammation phase Once a physiological barrier to blood loss (hemostasis) is established via formation and stabilization of a platelet-rich fibrin clot, the various biomolecules released from platelet



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granules and lysosomes can substantially influence the subsequent immune response and inflammation phases to protect the wound against infection, priming the wound niche for repair. For example, platelets activate (as well as partly release) complement factors, and combined with interleukin activation and TGF- β signaling, this regulates concentration gradient-mediated chemotaxis of neutrophils as well as lymphocytes and monocytes106. Other platelet-secreted molecules like IL-1 and TNF-α induce intracellular adhesion molecules in endothelial cells and allow neutrophils to adhere to their surface. These neutrophils begin to neutralize infectious organisms by direct ingestion (phagocytosis), degranulation and release of toxic substances or by using protease traps (e.g. neutrophil extracellular traps or NETs) which kill bacteria in the extracellular space98. As the inflammatory phase progresses, neutrophils are engulfed by macrophages which continue the process of phagocytosis108. Macrophages can function at a lower pH, have a longer lifespan and arrive at the wound site with a large reservoir of growth factors such as TGF-β, TGF-α, heparin binding epidermal growth factor, fibroblast growth factor (FGF), as well as, matrix remodeling enzymes like matrix metalloproteases (MMPs)

106,109,110

. Platelet components like CXCL4 contribute to

neutrophil recruitment and degranulation, as well as regulating macrophage recruitment and polarization from the pro-inflammatory state to the repair phenotype. Lymphocytes are next to arrive at the wound site and play a role in regulating wound healing via facilitation of matrix remodeling111.


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C.3. Platelet role in Proliferation phase As the primary dangers to the wound site are cleared by hemostasis and early-to-mid phases of inflammation, the wound healing process moves to the proliferation stage where the main goal is to repair the tissue defect via granulation tissue and synthesis of extracellular matrix (ECM) and vasculature. As platelets (and macrophages) release TGF-β and PDGF, they help fibroblasts to migrate towards the wound and proliferate causing the production of ECM components such as hyaluronan, proteoglycans, fibronectin and collagen112,113. As the matrix synthesis nears completion, angiogenic factors such as TGF-α, TGF-β, FGF, VEGF and PDGF, which are released during the coagulation phase and continue to be released during the inflammatory phase, regulate repopulation of the wound area with endothelial tissue. As they proliferate, endothelial cells undergo vasculogenesis and tube formation through the wound to restore vascular supply97,114. TNF-α, reportedly produced in part from neutrophils, macrophages, mast cells, platelets, endothelial cells and fibroblasts, can promote upregulation of proliferation-relevant integrins on cells and can also facilitate transformation of fibroblasts into myofibroblasts to aid wound contraction106,115,116. Inhibitory and regulatory factors such as angiostatin, interferon-inducible protein (IP-10) and steroids ensure optimum proliferation beyond which the cascade moves to the remodeling phase. These signaling molecules begin to downregulate growth factors, inhibiting motility and limiting recruitment to wound site106,117–119.



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C.4. Platelet role in Remodeling phase The final phase of wound healing consists of epithelialization, scar tissue formation and maturation. In this phase, the synthesis, degradation and remodeling of extracellular matrix and other tissue components (e.g scar tissue) proceed in a balance. MMPs remove matrix barriers by degrading the ECM, thereby allowing keratinocytes to migrate and MMPs (e.g. platelet-relevant MMP-2 and MMP-9) induce the release of ECM-bound factors such as VEGF and TNF-α120. TNF-α stimulates the release of IL-6 by fibroblasts, which along with TGF-β can upregulate the expression of tissue inhibitors of metalloproteinases (TIMPs) and tissue adhesion proteins121. As the activity of TIMPs increases, the MMPs lose their activity and the connective matrix shrinks to close the wound. This process is primarily mediated by PDGF, FGF and TGF- β, all of which can be partly derived from activated platelet granule secretions. TGF-β directs replacement of proteoglycans and fibronectin by collagen I, thereby helping the progression of matrix reconstruction121. PDGF and VEGF also contribute to wound remodeling via fibroblast regulation and extravasation of plasma proteins that form the support for epithelial and endothelial cells122. Macrophages and fibroblasts undergo apoptosis and recede from the wound site paving the way for reduced metabolic activity at the wound site and gradual maturation and structural integrity of scar tissue108,123–126.

D. Platelet-relevant Treatments in Wound Healing The plethora of treatments available to patients in need of wound care is as vast as the types of wounds clinically presented. These treatments range from ancient strategies like use of silver as an anti-infective agent (which is still used for such purposes), to


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more modern strategies like use of drug/growth factor-loaded biomaterials and advanced platelet-derived products. The following sections will discuss the different platforms and materials available for wound care/treatment, the promises and challenges of such strategies, and the potential of platelet-inspired biomaterials technologies in this area.

D.1. Traditional treatments and biomaterial-based approaches Ionized silver has been in use in the treatment of wounds for centuries owing to its ability to bind negatively charged particles such as proteins, DNA, RNA, and chloride ions and neutralizing microbial threats to the wound5. However, recent research into more complex wound treatments has resulted in the development of systems that combine two or more functions that aid wound healing. Some such systems include advanced dressing systems, scaffold or gel based delivery systems and particle based delivery systems. Although silver is also used in wound dressings, these systems have advanced to include antimicrobial, debridement and absorbent properties that allow them to better mimic the natural environment at the wound site5. Dressings made from foam, alginates, hydrocolloids, hydrogels etc. have gained popularity because they possess exudate absorbent properties as well as space filling characteristics127. PEG hydrogels, in addition to being used for their absorbent properties, have been explored as a modality with spatially and temporally regulated delivery. These hydrogels, as well as several other polymer platforms such as poly(lactide-co-glycolide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyurethane etc., can be modified to be responsive to chemical, biological or mechanical stimuli such that these cues can be used to deliver wound site relevant factors and drugs to speed up healing128,129. In addition to polymeric



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systems for wound site delivery, advances in nanotechnology have allowed the development of surfaces and scaffolds that can deliver drugs and growth factors to the wound along with possessing nanoscale features that can mimic natural cell properties4. This has led to the development of various different kinds of nanomaterials for regenerative medicine including surface modifications of biomaterial implants, nanoparticles for delivery of molecules, nanofibers for tissue scaffolds, and nanodevices4. Nanoscale delivery methods represent a new direction delivery systems are moving in where spatio-temporal control of payload release is at the forefront of device design. Nanoparticle-based therapies in development including silver, gold, polyacrylate, chitosan and lipid-based nanoparticles have also shown wound healing potential130.

Micro-patterned,

nano-sized

fiber

matrices

and

surface-modified

nanobioglass were shown to improve the efficiency and re-epithelialization of wound healing131. Several of these non-biological platforms such as nanoparticles and PEG hydrogels can be used to deliver proteins and enzymes to the wound site to affect specific wound healing processes.

D.2. Platelet-based derivatives In recent years, platelet gels, platelet rich plasma (PRP), platelet derived growth factors and other derivatives of platelets have emerged as interesting technologies to be utilized for their wound healing properties. Autologous platelet gels are prepared from a patient’s blood by first separating the platelet rich plasma from whole blood and then adding Calcium chloride and thrombin. Platelet gel has been demonstrated to be able to reduce pain and increase granulation tissue content and epithelialization in chronic wounds132. In another research, platelet rich fibrin matrix was shown to have significant


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potential in closing chronic venous leg ulcers133. Platelet gel (also called Platelet rich plasma (PRP) gel) has been demonstrated to be able to heal diabetic foot ulcers and complex wound with bone exposure134,135. Platelet rich plasma has also been shown to promote differentiation of human dermal fibroblasts into myofibroblasts as well as wound contraction136. Autologous platelets, in combination with leukocytes, have been shown to be potent in inducing wound healing in infected soft tissue injury137. Traumatic or surgical loss of finger tissue was also treated with autologous platelets to achieve favorable results138. In equine wound healing, activated platelet-rich plasma gel has been shown to accelerate epithelial differentiation and improve tissue regeneration and collagen organization139. Platelet derived products other than platelet gels have also proved effective in tackling wound healing. In one such example, quiescent platelets induced hemostasis, angiogenesis and wound healing in diabetic wounds140. In a systematic review, it was reported that in a majority of studies, platelet rich fibrin promoted soft tissue regeneration and wound healing in a variety of procedures141. While such platelet (and PRP)-based technologies are becoming attractive candidates in wound healing research and applications, it should be noted that these products vary widely in their composition and concentrations and currently lack proper clinical standardization aspects. It should also be noted that application of such platelet-based systems may provide advantages in ‘wound localized delivery’ but systemic administration of such products may pose off-target negative effects, similar to platelet hyperactivity associated complications in platelet transfusion and microvascular injury. Figure 5 shows examples of platelet treatment on in vitro and in vivo wound models,



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indicating that incubation with platelets augments healing mechanisms like blood vessel development and re-epithelialization.

In addition to whole platelets and their gel-based formulations, platelet derived growth factors and released biomolecules can also act as effective treatments in wound healing. For example, platelet rich clot releasate has been shown to be effective in remodeling the extracellular matrix as it can upregulate matrix metalloproteinase (MMP)-1 and type-1 collagen in fibroblasts142. Treatment with platelet derived growth factor (PDGF) has also been shown to stimulate healing of chronic wounds143. Due to storage and transfusion limitations of platelet and PRP gels, longer term techniques are currently being explored for application platelet-based wound healing products and treatments. For example, a trehalose based freeze drying method has been developed which allows outdated platelets to retain normal levels of PDGF-ββ and TGF-β1144. Freeze dried outdated platelets were shown to be able to promote endothelial cell differentiation at a level similar to that of freeze dried indated platelets and room temperature platelets. These freeze dried outdated platelets also helped accelerate wound closure better than controls145. Freeze dried platelets, in combination with vascular endothelial growth factor (VEGF), were shown to be capable of successfully closing wounds 90% faster than untreated wounds, representing a promising strategy for use of platelet derived products in the realm of injury treatment144. Vascular endothelial growth factor–A, platelet-derived growth factor-ββ and bone morphogenetic protein–2, when fused with a domain in placenta growth factor-2, were found to have enhanced affinity towards the regenerating ECM and promoted repair in rodent models of chronic wounds and bone defects146. Figure 6 shows examples of beneficial effects of


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platelet lysate derivatives (e.g. platelet lysate gel) on augmenting angiogenic and proliferative outcomes in wound healing models in vitro and in vivo.

D.3. Platelet-inspired biomaterials technologies Transfusions of whole blood and blood components (e.g. RBCs, platelets and freshfrozen plasma) are often used in treating heavy bleeding wounds, to promote rapid hemostasis as a precursor for subsequent inflammation and healing147,148. However, blood products, especially platelet concentrates, suffer from several logistical and functional challenges including limited availability, high risk of bacterial contamination, very short shelf-life and low portability, that has limited the widespread use of these products in transfusion and wound healing applications149,150. To resolve such challenges, several biomaterials-based strategies have focused on creating semisynthetic and synthetic mimics of platelets. Earlier approaches in this area focused on coating polymeric (e.g. polyacrylamide particles), liposomal, albumin or RBC particles with fibrinogen (Fg) or Fg-mimetic Arginine-Glycine-Aspartic Acid (RGD) peptides151–157. These semi-synthetic and synthetic platelet mimics were shown to be able to reduce bleeding in several animal injury models (i.e. promote effective hemostasis), but have not been explored further for delivery of platelet-relevant biomolecules to facilitate wound healing. In recent years, nanotechnology approaches have resulted in the development of several refined synthetic platelet designs, e.g. polylactic acid (PLA)based solid polymeric nanoparticles surface-decorated with Fg-relevant RGD peptides, poly-N-isopropylacrylamide-co-acrylic acid (pNIPAm-AAc) microgel particles surfacedecorated with fibrin-binding nanobodies, liposomal or polymeric particles surface-



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decorated

with

fibrinogen

γ-chain

dodecapeptide,

and

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liposomal or albumin

nanoparticles surface-decorated heteromutivalently with vWF-binding, collagen-binding and active platelet integrin GPIIb-IIIa-binding peptide ligands158–163. Figure 7 shows representative design schematics of various biomaterials-based semi-synthetic and synthetic mimics of platelets. These designs have all shown the capability to promote hemostasis in treating bleeding injuries to various extents, in several different animal models. Furthermore, some of these systems have been demonstrated to be amenable for utilization as platelet-inspired drug delivery platforms164–168. The potential for mimicking platelet granule content has shown promise in platforms such as those where platelet polyphosphate has been encapsulated in nanoparticles (granular nanoparticles and artificial dense granules) and tested for its ability to enhance coagulation and the results were encouraging169,170. Localizing polyP on gold nanoparticles was also shown to be effective in triggering the contact pathway for coagulation171. In addition to targeting primary hemostasis for enhanced coagulation, products such as PolySTAT have targeted secondary hemostasis and fibrin polymerization to enhance clot strength172. These reports have opened up the possibility to refine ‘synthetic platelet’ designs to incorporate some of platelet’s secretome and releasate components as payload for potential use as wound-healing technologies. For example, it can be envisaged that various ‘synthetic platelet’ technologies can be loaded with anti-infective agents as well as platelet-relevant growth factors (e.g. VEGF, PDGF etc.) to be released in a spatio-temporally controlled way at the site of hemostatic action, so as to facilitate wound healing mechanisms beyond hemostasis. In fact, the use of platelet lysates in conjunction with hydrogels and polysaccharide nanoparticles has proven to


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be

effective

in

improving

angiogenesis,

proliferation

of

endothelial

cells,

neovascularization as well as cartilage regeneration173–176. Tissue regeneration and blood vessel regeneration were also enhanced by gelatin hydrogel granules incorporating mixed PGFM and bFGF and by PDGF contained in a conjugated fibrin matrix177,178. Although these particulate drug delivery systems are not ‘synthetic platelet’ technologies by design, one can envisage adapting such delivery mechanisms and facilitating similar results using a synthetic platelet platform in an injury site-selective manner. Figure 8 shows examples of beneficial effects of platelet-relevant growth factor delivery in wound healing. Furthermore, synthetic platelet designs can be integrated within other biomaterialsbased matrix systems to create multi-component technologies to treat wounds and facilitate healing. Certain matrix systems have already been demonstrated for direct loading and delivery of platelet-relevant biomolecules. For example, a recombinant basic fibroblast growth factor loaded on a kind of absorbable collagen sponge system has been shown to reduce healing time as well as wound closure time179. Tunable hydrogels have also been shown to mediate delivery of cell-secreted growth factors and cytokines to promote wound healing180. In another interesting aspect of wound healing research, recent studies have shown that controlled delivery of cytokines (e.g. interferon gamma, IL-4 etc.) could be used to modulate macrophage conversion to promote tissue regeneration181. In another report, PEG-Fibrin gels were designed to express smooth muscle cell markers (such as α-smooth muscle actin, platelet-derived growth factor receptor-β, NG2 proteoglycan, and angiopoietin-1) that could facilitate formation of vascular structures in healing wounds182. Certain polymeric systems have recently been



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reported in the context of augmenting clot strength or promoting embolization of blood vessels. For example, a co-polymer made from hydroxyethyl methacrylate (HEMA) and N-hydroxysuccinimide methacrylate (NHSMA), (p(HEMA-co-NHSMA)), has been modified with fibrin cross-linking peptides and intravenous administration of this polymer has been shown to augment fibrin strength and stability at the bleeding site in a small animal model172. In another report, a shear thinning polymer composite made of gelatin and silica nanoparticles was shown to be able to promote embolization of bleeding vessel183. In yet another report, self-expandable sponges made of wood pulp coated with chitosan were shown to be capable of administered intra-cavitarily in open wounds to staunch bleeding via stypsis and absorption184. Polymeric biomaterials based technologies have also been reported where blood components (e.g. PRP) can be encapsulated within enzyme-degradable hydrogel matrices to allow sustained degradation, release, activation and secretion of such blood components to modulate wound

healing mechanisms185,186. These are examples of

polymer systems

administered topically, intracavitarily or intravascularly, to render direct contact with injury or bleeding sites and hence such polymers can be potentially loaded directly with various biomolecules that promote wound healing or with platelet-mimetic drug delivery particles that can release biomolecules to modulate healing mechanisms over time.

E. Discussion Following an injury, a complex cascade of intracellular, intercellular and extracellular mechanisms occur in a very regulated fashion to allow hemostasis, inflammation, proliferation and remodeling phases of wound healing. Blood platelets play a central


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role in these phases by virtue of their primary role in hemostasis, as well as, their sustained role in influencing inflammatory and immune responses. As a result, plateletbased products, such as platelet-rich plasma, platelet gel, platelet-rich fibrin and platelet eye drops are becoming pre-clinically evaluated and clinically attractive technologies to prompt wound healing and tissue regeneration15,187–189. However, there is a wide variability in the preparation, composition and concentrations of these products, reported in the various studies, which makes standardized correlations difficult. In recent years, a large number of clinical trials have shown the promise of these plateletbased products in wound healing and tissue regeneration, but at the same time, the absence of accepted standards for the preparation of these various platelet products have somewhat undermined the clinically significant broad applicability of such products. For example, in review and meta-analyses of clinical studies, some studies have shown beneficial effect of PRP-based therapies in plantar fasciitis small-tomoderate rotator cuff tears, tendinopathy, and musculo-skeletal injuries, while some studies have shown that PRP therapy does not provide additional benefit in certain injuries like acute hamstring injury190–194. These analyses essentially indicate that the variations in clinical outcomes are possibly a result of variation in PRP gel preparation procedures, activation and administration times. They also suggest that pre-activating all platelets in PRP gel and thereby essentially maximizing platelet secretome production prior to injury site administration may not be very efficacious, since the sequential temporal regulation of the effects of various secreted components may be lost in this approach. Rigorous pre-clinical studies should focus on resolving these issues and variabilities in the context of spatio-temporal modulation of effects. Building



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on that, efficaciously planned multicenter clinical studies with sufficiently large patient volume are needed to further emphasize the utility of such platelet-based therapies in wound healing.

In parallel to such natural platelet-based products, a robust volume of research is currently being directed at leveraging and mimicking the mechanisms and secretions of platelets using a variety of biomaterial platforms, including synthetic platelet mimics and platelet-relevant biomolecule releasing delivery platforms. The importance of platelet secretome, including pro-coagulant molecules, growth factors, cytokines and enzymes, in hemostatic and inflammatory phases of wound healing is undeniable. Biomaterialsbased approaches to leverage the action of these biomolecules should focus not only on wound site-selective delivery but also on the spatio-temporal control of the release patterns, since various biomolecules are required to play sequential roles through the time frame of wound healing. Biomaterials based controlled drug delivery systems that can regulate the concerted mechanisms through spatio-temporally controlled release mechanisms may provide unique advantages in mimicking and integrating the hemostatic and secretory roles of platelets in wound healing and tissue regeneration. Additionally, bio-hybrid systems can be developed by combining PRP-based components with synthetic biomaterials based platforms, as evident from recent reports on pre-clinical studies195,196. Continued research in this area can lead to unique biomaterials-based technologies that leverage platelet’s inherent wound healing components and mechanisms to augment tissue repair and regeneration.


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Acknowledgement UDS Sekhon and A Sen Gupta are supported by the grant 5R01 HL121212 (PI: Sen Gupta) from the National Institutes of Health. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figure legends Figure 1. (A) Schematic representation of platelet surface receptor molecules and their corresponding ligands (or substrates); Interaction between these systems is paramount for hemostatic function of platelets; (B) Representative Scanning Electron Microscopy (SEM) images of quiescent (resting) ‘discoid’ platelet(s), activating ‘stellate’ platelet with pseudopodal protrusions and fully active ‘spread’ platelet that has possible released its granule contents. For capturing SEM images, prostaglandin E1 (PGE1)-treated quiescent platelets or Calcium (Ca++)-treated semi-active platelets or ADP-treated fully active platelets were adhered by incubation onto fibrinogen-coated glass coverslips; adhered quiescent or active platelets were fixed in 2.5% glutaraldehyde for 1 h at 4oC, then subjected to progressive dehydration with graded series of ethanol solutions followed by critical point drying in liquid CO2, platelet-adhered coverslips were attached to SEM sample stubs and sputter coated with platinum, and imaging was done with a Hitachi S-4500 field emission electron microscope with an accelerating voltage of 5 kV.

Figure 2. Platelet granules and cytosolic entities, along with granule and cytosolic contents secreted upon platelet activation; [A] shows representative electron



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microscopic image of a platelet exhibiting the various cytosolic and granule ultrastructures, while [B] shows a cartoon representation of these structures; [C] shows a representative list of biomolecules secreted from various platelet granules and lysosomes that play important roles in various phases of wound healing. Reproduced with permission from ref. 24 Copyright 2013 Fitch-Tewfik and Flaumenhaft and ref. 27 Copyright 1993 Elsevier.

Figure 3. Schematic representation of four phases of wound healing and tissue repair, namely hemostasis, inflammation, proliferation and remodeling phases where platelets and platelet secretome play important mechanistic roles in augmenting coagulation, modulating recruitment of neutrophils and monocytes, influencing monocyte-tomacrophage transformations and macrophage polarization, promoting angiogenesis and fibroblast proliferation, and facilitating matrix organization and remodeling towards tissue repair. Modified with permission from ref. 197 Copyright 2011 McGraw Hill

Education.

Figure 4. Schematic representation of platelet’s primary role in hemostasis; platelets can adhere to wound site by binding to collagen and von Willebrand Factor (vWF), activate as a result of these adhesion mechanisms as well as due to action of locally generated thrombin (e.g. via Tissue Factor pathway) and ADP, aggregate with other activated platelets via fibrinogen (Fg) binding to stimulated integrin GPIIb-IIIa on platelet surface, facilitate coagulation pathways via co-localization of coagulation factors on active platelet surface to amplify thrombin generation and conversion of Fg into fibrin,


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and release various granule and lysosomal contents to subsequently influence successive phases of immune response, inflammation, angiogenesis, cellular proliferation and matrix remodeling in wound healing and tissue repair.

Figure 5. Beneficial effect of platelet incubation on wound healing components in vitro and in vivo; [A] demonstrates dose dependent effect of incubating washed platelets (or platelet-relevant growth factors) on angiogenesis in the rat aortic ring model, with [A1] showing microscopic images of (1) negative control, (2) 0.2 × 105/µl platelets, (3) 2 × 105/µl platelets, (4) 5 × 105/µl platelets, (5) 1 × 106/µl platelets and (6) VEGF+FGF (50 ng/ml of each) and [A2] shows corresponding quantitative results from these studies; [B] demonstrates dose dependent effect of incubating platelet releasate (obtained from an increasing concentration of platelets) on angiogenesis in the rat aortic ring model, with [B1] showing microscopic images of (1) negative control, (2) releasate from 0.2 × 105/µl platelets, (3) releasate from 2 × 105/µl platelets, (4) releasate from 5 × 105/µl platelets, and (5) releasate from 1 × 106/µl platelets, and [B2] showing corresponding quantitative results from these studies; [C] demonstrates different levels of wound closure at day 9 after injury upon incubation with multiple doses of fresh platelet-rich-plasma (PRP-m), versus single or multiple doses of Trehalose-treated lyophilized reconstituted platelets (TLP and TLP-m respectively), versus multiple doses of VEGF (VEGF-m) or no treatment (NT), where [C1] shows the microscopic images of these incubations on wound closure and [C2] shows the amount of contraction, re-epithelialization and raw surface of the wound remaining after day 9, indicating improved outcomes of wound healing for PRP treated ands VEGF treated wounds. Reproduced with permission from



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ref. 45. Copyright 2004 Oxford University Press and ref. 144. Copyright 2007 John Wiley & Sons.

Figure 6. Beneficial effect of platelet-derived lysates and gels on wound healing components in vitro and in vivo; [A] shows tissues obtained 79 days after wounding and stained with Masson’s trichrome stain to analyze collagen amount and arrangement; [A1] and [A3] show microscopic image of the tissue (scale bar 100 µm) while [A2] and [A4] show tissue zoomed in (scale bar 200 µm), indicating that the PRP lysate treated wounds have well-organized collagen bundles oriented parallel to the overlying epithelia (characteristic of mature granulation tissue) while the untreated tissue has randomly organized collagen bundles (characteristic of immature granulation tissue); [B] demonstrates effect of platelet lysate gel (PL Gel) on angiogenic sprouting, with [B1] showing the method for PL Gel preparation, [B2] and [B3] showing fluorescence and scanning electron microscopy images respectively of clot formation with dense fibrin network after PL Gel application, and, [B4] and [B5] showing improved effect of PL Gel incubation on endothelial-specific sprouting in HUVECs when compared to incubation with only fibrin gel (Fib Gel); [C] demostrates representative results from experiments of hind limb ischemia reperfusion in NOD-SCID mice, where mice were treated with mesenchymal stem cells

(MSCs) in

PL

Gel showed

significantly improved

neovascularization and blood flow restoration compared to treatment with control groups, as analyzed by [C1] laser Doppler image-assisted [C2] quantification of limb perfusion at 1 and 8 days. Reproduced with permission from ref. 139. Copyright 2003


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Elsevier, ref. 144 Copyright 2007 John Wiley & Sons and ref. 157 Copyright 2016 John Wiley & Sons.

Figure 7. Schematic representation of various biomaterials-based approaches in designing semi-synthetic or fully synthetic platelet analogs, by mimicking and amplifying platelet’s adhesive and aggregatory mechanisms that primarily promote hemostasis.

Figure 8. Beneficial effects of delivering platelet secretome-relevant biomolecules and growth factors delivered from biomaterials-based matrices and scaffolds; [A] and [B] show immunohistological images and corresponding quantitative data analysis of in vivo angiogenic effects of a gelatin hydrogel containing PRP growth factor mixture (PGFM) with and without bFGF administered intramuscularly into a mouse leg ischemia model (results shown are for 1 week after treatment); PGFM, bFGF and PGFM + bFGF conditions all showed higher extent of blood vessel development compared to controls (PPP: platelet poor plasma), and PGFM + bFGF group showed higher α-SMA-positive blood vessels compared to ‘PGFM only’ or ‘bFGF only’ treatment groups (SMA: smooth muscle actin); [C] shows immunohistochemical evaluation of functional angiogenesis (microscopic images and corresponding quantitative analysis) in rodent epigastric flap model, where higher ratios of mature functional blood vessels (SMA+/vWF+) were found for treatment groups of Transglutaminase-crosslinked PDGF-AB chain (TG-PDGF.AB) in comparison to the control and fibrin sealant (FS) groups in the intradermal layer. Reproduced with permission from ref. 177. Copyright 2016 Elsevier and ref. 178. Copyright 2012 Elsevier.



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References 1. 2. 3.

4.

5. 6. 7. 8. 9. 10. 11.

12.

13.

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Outlook. Biomed Res Int. 2015;2015:1-24. doi:10.1155/2015/846045. 197. Prentice, W. Tissue response to Injury, Chapter 10 Figure 10-2, page 267, In: Principles of Athletic Training: A Competency-Based Approach, 15e, 2011; McGraw-Hill Education Material. ISBN: 9780078022647



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Figure 1. (A) Schematic representation of platelet surface receptor molecules and their corresponding ligands (or substrates); Interaction between these systems is paramount for hemostatic function of platelets; (B) Representative Scanning Electron Microscopy (SEM) images of quiescent (resting) ‘discoid’ platelet(s), activating ‘stellate’ platelet with pseudopodal protrusions and fully active ‘spread’ platelet that has possible released its granule contents. For capturing SEM images, prostaglandin E1 (PGE1)-treated quiescent platelets or Calcium (Ca++)-treated semi-active platelets or ADP-treated fully active platelets were adhered by incubation onto fibrinogen-coated glass coverslips; adhered quiescent or active platelets were fixed in 2.5% glutaraldehyde for 1 h at 4oC, then subjected to progressive dehydration with graded series of ethanol solutions followed by critical point drying in liquid CO2, platelet-adhered coverslips were attached to SEM sample stubs and sputter coated with platinum, and imaging was done with a Hitachi S4500 field emission electron microscope with an accelerating voltage of 5 kV. 99x116mm (300 x 300 DPI)


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Figure 2. Platelet granules and cytosolic entities, along with granule and cytosolic contents secreted upon platelet activation; [A] shows representative electron microscopic image of a platelet exhibiting the various cytosolic and granule ultrastructures, while [B] shows a cartoon representation of these structures; [C] shows a representative list of biomolecules secreted from various platelet granules and lysosomes that play important roles in various phases of wound healing. Reproduced with permission from ref. 24 Copyright 2013 Fitch-Tewfik and Flaumenhaft and ref. 27 Copyright 1993 Elsevier. 111x123mm (300 x 300 DPI)


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Figure 3. Schematic representation of four phases of wound healing and tissue repair, namely hemostasis, inflammation, proliferation and remodeling phases where platelets and platelet secretome play important mechanistic roles in augmenting coagulation, modulating recruitment of neutrophils and monocytes, influencing monocyte-to-macrophage transformations and macrophage polarization, promoting angiogenesis and fibroblast proliferation, and facilitating matrix organization and remodeling towards tissue repair. Modified with permission from ref. 197 Copyright 2011 McGraw Hill Education. 84x71mm (300 x 300 DPI)



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Figure 4. Schematic representation of platelet’s primary role in hemostasis; platelets can adhere to wound site by binding to collagen and von Willebrand Factor (vWF), activate as a result of these adhesion mechanisms as well as due to action of locally generated thrombin (e.g. via Tissue Factor pathway) and ADP, aggregate with other activated platelets via fibrinogen (Fg) binding to stimulated integrin GPIIb-IIIa on platelet surface, facilitate coagulation pathways via co-localization of coagulation factors on active platelet surface to amplify thrombin generation and conversion of Fg into fibrin, and release various granule and lysosomal contents to subsequently influence successive phases of immune response, inflammation, angiogenesis, cellular proliferation and matrix remodeling in wound healing and tissue repair. 48x23mm (300 x 300 DPI)


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Figure 5. Beneficial effect of platelet incubation on wound healing components in vitro and in vivo; [A] demonstrates dose dependent effect of incubating washed platelets (or platelet-relevant growth factors) on angiogenesis in the rat aortic ring model, with [A1] showing microscopic images of (1) negative control, (2) 0.2 × 105/µl platelets, (3) 2 × 105/µl platelets, (4) 5 × 105/µl platelets, (5) 1 × 106/µl platelets and (6) VEGF+FGF (50 ng/ml of each) and [A2] shows corresponding quantitative results from these studies; [B] demonstrates dose dependent effect of incubating platelet releasate (obtained from an increasing concentration of platelets) on angiogenesis in the rat aortic ring model, with [B1] showing microscopic images of (1) negative control, (2) releasate from 0.2 × 105/µl platelets, (3) releasate from 2 × 105/µl platelets, (4) releasate from 5 × 105/µl platelets, and (5) releasate from 1 × 106/µl platelets, and [B2] showing corresponding quantitative results from these studies; [C] demonstrates different levels of wound closure at day 9 after injury upon incubation with multiple doses of fresh platelet-rich-plasma (PRP-m), versus single or multiple doses of Trehalose-treated lyophilized reconstituted platelets (TLP and TLP-m respectively), versus multiple doses of VEGF (VEGF-m) or no treatment (NT), where [C1] shows the microscopic images of these incubations on wound closure and [C2] shows the amount of contraction, reepithelialization and raw surface of the wound remaining after day 9, indicating improved outcomes of wound healing for PRP treated ands VEGF treated wounds. Reproduced with permission from ref. 45. Copyright 2004 Oxford University Press and ref. 144. Copyright 2007 John Wiley & Sons. 99x101mm (300 x 300 DPI)



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Figure 6. Beneficial effect of platelet-derived lysates and gels on wound healing components in vitro and in vivo; [A] shows tissues obtained 79 days after wounding and stained with Masson’s trichrome stain to analyze collagen amount and arrangement; [A1] and [A3] show microscopic image of the tissue (scale bar 100 µm) while [A2] and [A4] show tissue zoomed in (scale bar 200 µm), indicating that the PRP lysate treated wounds have well-organized collagen bundles oriented parallel to the overlying epithelia (characteristic of mature granulation tissue) while the untreated tissue has randomly organized collagen bundles (characteristic of immature granulation tissue); [B] demonstrates effect of platelet lysate gel (PL Gel) on angiogenic sprouting, with [B1] showing the method for PL Gel preparation, [B2] and [B3] showing fluorescence and scanning electron microscopy images respectively of clot formation with dense fibrin network after PL Gel application, and, [B4] and [B5] showing improved effect of PL Gel incubation on endothelial-specific sprouting in HUVECs when compared to incubation with only fibrin gel (Fib Gel); [C] demostrates representative results from experiments of hind limb ischemia reperfusion in NOD-SCID mice, where mice were treated with mesenchymal stem cells (MSCs) in PL Gel showed significantly improved neovascularization and blood flow restoration compared to treatment with control groups, as analyzed by [C1] laser Doppler image-assisted [C2] quantification of limb perfusion at 1 and 8 days. Reproduced with permission from ref. 139. Copyright 2003 Elsevier, ref. 144 Copyright 2007 John Wiley & Sons and ref. 157 Copyright 2016 John Wiley & Sons. 150x86mm (300 x 300 DPI)



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Figure 7. Schematic representation of various biomaterials-based approaches in designing semi-synthetic or fully synthetic platelet analogs, by mimicking and amplifying platelet’s adhesive and aggregatory mechanisms that primarily promote hemostasis. 99x94mm (300 x 300 DPI)


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Figure 8. Beneficial effects of delivering platelet secretome-relevant biomolecules and growth factors delivered from biomaterials-based matrices and scaffolds; [A] and [B] show immunohistological images and corresponding quantitative data analysis of in vivo angiogenic effects of a gelatin hydrogel containing PRP growth factor mixture (PGFM) with and without bFGF administered intramuscularly into a mouse leg ischemia model (results shown are for 1 week after treatment); PGFM, bFGF and PGFM + bFGF conditions all showed higher extent of blood vessel development compared to controls (PPP: platelet poor plasma), and PGFM + bFGF group showed higher α-SMA-positive blood vessels compared to ‘PGFM only’ or ‘bFGF only’ treatment groups (SMA: smooth muscle actin); [C] shows immunohistochemical evaluation of functional angiogenesis (microscopic images and corresponding quantitative analysis) in rodent epigastric flap model, where higher ratios of mature functional blood vessels (SMA+/vWF+) were found for treatment groups of Transglutaminase-crosslinked PDGF-AB chain (TG-PDGF.AB) in comparison to the control and fibrin sealant (FS) groups in the intradermal layer. Reproduced with permission from ref. 177. Copyright 2016 Elsevier and ref. 178. Copyright 2012 Elsevier.



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99x125mm (300 x 300 DPI)


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Graphical Abstract 50x41mm (300 x 300 DPI)


Platelets and Platelet-Inspired Biomaterials Technologies in Wound

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