Wound Healing Society

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In Vitro Study Of An Autologous Leukocyte And Platelet-rich Fibrin Patch For Managing Diabetic Foot Ulcers
Jonas D. Schmidt, PhD1, Rasmus Lundquist2.
1University of Copenhagen, Copenhagen, Denmark, 2Reapplix ApS, Birkerød, Denmark.

AimTo investigate immune responsiveness and growth factor release of an autologous leukocyte-and-platelet-rich fibrin patch (APFP) for treating chronic wounds. MethodsAPFPs were prepared from healthy donors as described in Lundquist, et al20131. In short, 18 ml blood was filled into a vacuumed device2and processed for 20 min in an automated 2-step program. The resulting 3-layered patch consist of fibrin, platelets and leukocytes. APFP were analyzed for immune cells by histopathology and flowcytometry. Cytokine and growth factor production was analyzed in culture supernatants after in vitro stimulation with: lipopolysaccharide (LPS), IL-4/IL-13 and Chronic Wound Fluid (CWF) and cytokine and growth factor production was assessed by ELISA. APFP culture supernatants were added (10%) to keratinocyte cell line (HaCaT) cultures for 18 hours and proliferation was assessed by MTT assay, and migration by scratch assay. A skin explant model (approval number: H-17041884) was used to evaluate APFP effect on epithelization. ResultsWe show that APFP contain intact monocytes, T cells and B cells. APFP responded with production of IL-1beta, IL-6, IL-10 and IL-1Ra to LPS and CWF in vitro stimulation (48 hours), respectively. In contrast, IL-4/13 stimulation resulted in exclusive IL-1Ra production. Furthermore, supernatants from APFP cultures induced proliferation and migration in HaCaT cells and skin explant wounds. Topical application of APFP biopsies on skin explant wounds showed migration of the advancing epithelial tongue. ConclusionsWe find that APFP can respond and adapt appropriately in the management of chronic wounds by delivering immune cells, cytokines and growth factors.

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