Activation of platelet-rich plasma by pulse electric fields: Voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin.

Bogdan Neculaes,Emma E Forde,Alan D Michelson,Andrew L Frelinger,Anja J Gerrits,Thomas Gremmel,Sabrina L Carmichael,Steven Klopman,Pablo Garcia De Frutos

doi:10.1371/journal.pone.0249209

Abstract

Activated platelet-rich plasma (PRP) has been used in the clinical settings of wound healing and regenerative medicine, with activation typically induced by the addition of bovine thrombin. To eliminate issues with availability, cost and potential side effects associated with bovine thrombin, ex vivo PRP activation using pulse electric fields (PEF) has been proposed and demonstrated. The present study characterizes the effect of PEF voltage and pulse width, in combination with a range of calcium concentrations, on clot formation, growth factor release, and serotonin (5-HT) release from dense granules. The main findings are: 1) increasing calcium concentrations with most PEF conditions leads to increased levels of PDGF and 5-HT release; 2) whether EGF levels increase or decrease with increasing calcium concentration depends on the specific PEF parameters; 3) the pattern of PDGF and EGF levels in supernatants suggest that these molecules are localized differently within platelets; 4) significant levels of PDGF, EGF, and 5-HT can be released without inducing clot formation or hemoglobin release. In conclusion, voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin from PEF-activated PRP. Because growth factor requirements vary for different types of wounds and for wounds at different stages of healing, the unique balance of factors in supernatants of PEF-activated PRP may provide more clinically advantageous than the current standard of bovine thrombin-activated PRP.

Highlights

Platelet rich plasma has been explored for various clinical applications, leveraging the growth factors and proteins released by platelets upon activation [1,2,3,4,5,6], Promotion of wound healing by clinically administered platelet-rich plasma (PRP) includes several steps: blood draw from the patient; PRP separation from whole blood; activation–typically with bovine thrombin; topical application of the activated PRP on the wound
The present study characterizes the effect of pulse electric fields (PEF) parameters, in combination with a range of calcium concentrations, on clot formation, hemoglobin release, growth factor release, and dense granule serotonin release
The main findings are: 1) increasing calcium concentrations with most PEF conditions leads to increased levels of PDGF and 5-HT release; 2) whether EGF levels increase or decrease with increasing calcium concentration depends on the PEF condition; 3) the pattern of PDGF and EGF levels in supernatants suggest that these molecules are localized differently within platelets; 4) significant levels of PDGF, EGF, and 5-HT can be released without inducing clot formation or hemoglobin release

Summary

Introduction

Platelet rich plasma has been explored for various clinical applications, leveraging the growth factors and proteins released by platelets upon activation [1,2,3,4,5,6], Promotion of wound healing by clinically administered platelet-rich plasma (PRP) includes several steps: blood draw from the patient; PRP separation from whole blood; activation–typically with bovine thrombin ( there is no clinical standard for bovine thrombin activation); topical application of the activated PRP on the wound. For specific PRP applications, the activation step is omitted, and non-activated PRP is directly injected at the site of the injury. These workflows attempt to harvest the effects on the wound healing cascade of growth factors released from platelets. Subsequent research discovered that PEF treatment of PRP enables growth factor release with or without clotting [13]–a unique feature that adds additional clinical functionality compared to the use of bovine thrombin. One could envision PEF-induced growth factor release and clotting of PRP for use in topical applications, and PEF-induced growth factor release without clotting of PRP for use in injections to accelerate the healing of injured tendons, ligaments, muscles and joints

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Conclusion