In vitro investigation of the therapeutic potential of canine platelet lysate in wound healing

Abstract

Wound healing is a complex multicellular process characterized by different phases that are mediated by a combination of cascades, such as mediators and cellular components, and requires multiple cytokines and growth factors. During the inflammatory phase of the wound healing, platelets influx to the injury site to form the hemostatic plug and release cytokines and growth factors through degranulation in the wound area. These factors promote the migration and chemotaxis of inflammatory cells into the wound, enhance tissue regeneration, and synthesis and deposition of extracellular matrix. Recent efforts have been focused on the development of platelet-derived products, such as platelet lysate as an economical source of growth factors, cytokines, chemokines, and osteoconductive proteins for wound healing and tissue regeneration. Our first aim was to evaluate how different methods of preparation used to manufacture various platelet-derived formulations affect the growth and cytokine concentration of the final product. For our studies, we generated and assessed different manual methods to prepare canine platelet lysate with variable contents of leukocytes, plasma, and heat-sensitive proteins. Double centrifugation of whole blood was used to generate platelet concentrates, a pure (PC, leukocyte poor) and a leukocyte rich platelet concentrate (LPC), while a portion of both products generated with each method underwent plasma removal. Following that, platelet lysate was generated via freeze-thaw cycles and a portion of the generated platelet lysate underwent complement inactivation via heat treatment. Growth factors associated with the healing process were quantified using enzyme-linked immunosorbent assay (ELISA). We showed that platelet-derived growth factor (PDGF) was significantly higher in platelet lysate that was plasma depleted, whereas vascular endothelial growth factor (VEGF) was significantly higher in heat-treated lysate groups. Furthermore, VEGF concentration in plasma-depleted leukocyte rich platelet lysate was significantly higher than plasma-depleted pure platelet lysate. Transforming necrosis factor-α (TNF-α) concentrations were overall very low, but significantly increased following plasma depletion. These results support that platelet lysate preparation and processing can affect growth factors and cytokine release. Our second goal was to investigate how different concentrations of various formulations of canine PL, with or without plasma proteins and heat-sensitive proteins, affect major activities of the regenerative process, such as the viability, chemotaxis, and migration of canine keratinocytes in vitro. We found that the keratinocytes maintained viability for up to 48 hours following the addition of various lysate formulations while a cytotoxic effect was not encountered. In addition, we found that lysates rich in plasma and content from platelets are essential for an improved chemotactic and migration activity of canine keratinocytes while plasma components alone do not seem to have a chemoattractant effect on keratinocytes. Our findings suggest that different formulations of canine platelet lysate affect the growth factor, cytokine release, and in vitro tissue regeneration activities, such as viability, chemotaxis, and scratch closure rate of canine keratinocytes. In conclusion, our study further supports the therapeutical potential of platelet lysate for the therapeutic management of canine wounds, as it might enhance tissue regeneration with a shortened recovery time.