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Pharmacological Preconditioning of an Ischemic Composite Tissue Using Platelet-Derived Extracellular Vesicles Delivered via a Hyaluronic Acid Hydrogel
Aida K. Sarcon, MD
1; Rou Wan, MD
1; Ramona Reisdorf, B.S
1; Julie F. Jacobs, D.V.M
1; Omar A. Selim, M.B.B.C.h, M.S
1; Elena Millesi, MD
1; Felicia D. Boynton, D.V.M
1; Anne Gingery, Ph.D.
1; Atta Behfar, MD, PhD
1; Chunfeng Zhao, MD
1; Steve L. Moran, M.D.
21Mayo Clinic, Rochester, MN; 2Plastic and Reconstructive Surgery, Mayo Clinic, Rochester, MN
Introduction: Pharmacological preconditioning may prime replanted digits/limbs against ischemia-reperfusion injury (IRI). We recently validated a novel IRI microsurgical composite tissue model via a rodent's biceps femoris musculocutaneous flap. We are now investigating the efficacy of platelet-derived extracellular vesicles (EVs) delivered via Hyaluronic Acid (HA) in our microsurgical composite tissue IRI model. We hypothesized that HA would serve as a suitable carrier to deliver EVs and that HA-EV-treated flaps would have less tissue injury.
Methods: 15 male rats (5 per group) were allocated to either HA-EVs, HA-alone, or no-treatment groups. All had the microsurgical IRI technique. Ischemia was induced with pedicle clamping (3 hours) and reperfusion by unclamping. HA (Lifecore Biomedical©) or platelet-derived EVs (moderate dose, Rion™, LLC) in HA were injected 30-minutes before reperfusion in skin/muscle (0.8mL total). Laser-speckle was used for perfusion characterization. The postop-perfusion unit (PU) was represented as a flap blood flow ratio (FBFR) compared to the limb's baseline. The serum and tissue were evaluated for injury. CM-Vybrant DiI was used to tag platelet-derived EVs to assess their retention. Protein release of platelet-derived EVs was performed in HA.
Results: Clamping reduced and unclamping increased perfusion (p<0.0001, Fig 1A). Untreated flaps were more ischemic at D0 (FBFR 0.8 vs 1.2 in treated, p=0.0231). Over time, the untreated flaps became more hyperemic compared to treated flaps (p=0.0005, Fig 1B). The HA-EV group had the lowest creatinine kinase (CK) and lactate dehydrogenase (LDH, Fig 1C) and qualitatively less myonecrosis (arrows, Fig 1D). CM-DiI-stained EVs were retained in the skin/muscle samples after 72 hours of reperfusion (N=1; arrows, Fig 1E). There was a dose-dependent relationship between the concentration of platelet-derived EVs and the rate of protein released. There was a gradual increase in the protein released over time (Fig 1F).
Conclusions: Pharmacological preconditioning with either HA or HA-EVs may result in a more stable perfusion pattern when compared to an untreated composite tissue following IRI. Preliminary data showed a trend of lower serum CK/LDH and less myonecrosis in HA-EV-treated flaps suggestive of reduced skeletal muscle injury. Our release assay showed gradual protein release over time (dose-dependent fashion). This suggests that HA may be a suitable carrier for EVs. Further validation and dose-optimization may be necessary to investigate the benefits of EVs in a composite tissue following IRI.
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