(LR-031) Pharmacological Preconditioning of an Ischemic Composite Tissue Using Platelet-derived Extracellular Vesicles Delivered via a Hyaluronic Acid Hydrogel
Introduction: Pharmacological preconditioning may prime flaps or 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 now investigate the efficacy of platelet-derived extracellular vehicles (EVs) delivered via Hyaluronic Acid (HA). We hypothesized that HA would serve as a suitable carrier for EVs and that HA-EV-treated flaps would have less tissue injury
Methods: 20 male rats were allocated to HA-EV20%, HA-EV50%, HA-alone, or no-treatment (5 per group). Ischemia was induced with pedicle clamping (3 hours) and reperfusion by unclamping. HA (Lifecore Biomedical) or platelet-derived EVs (moderate dose 20% or high dose 50%, Rion, LLC) in HA were injected 30-minutes before reperfusion in skin/muscle. 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). Over time the untreated flaps were hyperemic from POD0 to 2 (p=0.017). There were no differences in FBFR overtime in treated flaps. HA-EV50% had significantly higher inducible nitric oxide gene expression than no treatment (Fold 51.4 vs 1.4, p=0.039). HA-EV50% had significantly lower serum creatinine kinase compared to no treatment (101 vs 407 U/L, p=0.016,). Preliminary histology showed less myonecrosis with HA-EV20% (13%), HAEV50% (17.7%) compared to HA alone (39.2%) or no treatment (31.7%, Fig1 D). CM-DiI stained EVs showed retention in the skin/muscle samples after 72 hours of reperfusion (arrows, Fig1 E). 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.
Discussion: Pharmacological preconditioning with either HA-alone or HA-EVs may result in a more stable perfusion pattern versus no treatment of IRI. Preliminary data showed a trend of higher inducible nitric oxide synthase in HA-EV50% treated flaps. Ther was lower serum injury markers and less histological evidence of myonecrosis in HA-EV-treated flaps. 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 experimentation are warranted to evaluate therapeutic differences between moderate and high dose of EVs in composite tissue IRI .
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