Abstract
BackgroundActivation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization).AimsTo determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology.MethodsConcentrated normal donor PRP was activated by 5 μsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM).ResultsTime to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4–8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release.ConclusionPulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.
Highlights
Platelet secretory granules contain large amounts of proteins and growth factors which are known to have various beneficial effects on wound healing such as angiogenesis and tissue regeneration [1,2]
Time to initial clot formation was shorter with thrombin (
Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing
Summary
Platelet secretory granules contain large amounts of proteins and growth factors which are known to have various beneficial effects on wound healing such as angiogenesis and tissue regeneration [1,2]. Because wounds differ based on the type of injury (e.g., diabetic ulcer [6], burn [7]) and the stage of healing (e.g., granulation, angiogenesis, contraction, epithelialization [8,9]), and growth factors differ in their ability to influence these processes, it this unclear whether the balance of growth factors released by standard methods of platelet activation is optimal for all types and stages of wounds. In addition to platelet alpha granules, dense granules, lysosomes and endosomes, a new type of platelet granule has been more recently described by Thon et al [11] These so-called T-granules are unique electron-dense, membrane-delimited intracellular compartments in platelets and co-localize with the toll-like receptor (TLR) 9 and protein disulfide isomerase (PDI), making these proteins markers for T-granules. Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization)
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