脉冲电场可调活化治疗性富血小板血浆：对血栓形成、生长因子释放和血小板形态的差异影响。
Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology.
机构信息
Center for Platelet Research Studies, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, Massachusetts, United States of America.
GE Global Research Center, Niskayuna, New York, United States of America.
出版信息
PLoS One. 2018 Sep 26;13(9):e0203557. doi: 10.1371/journal.pone.0203557. eCollection 2018.
BACKGROUND
Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization).
AIMS
To determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology.
METHODS
Concentrated normal donor PRP was activated by 5 μsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM).
RESULTS
Time to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4-8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release.
CONCLUSION
Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.
背景
通过脉冲电场 (PEF) 激活富含血小板的血浆 (PRP) 会释放促进伤口愈合的生长因子（例如，用于肉芽形成的 PDGF、VEGF，用于上皮化的 EGF）。
目的
确定 PEF 激活治疗性 PRP 后的以下情况：1）血小板凝胶强度；2）释放的生长因子谱；3）α-和 T-颗粒释放；4）血小板形态。
方法
使用 5 μsec（长）单极脉冲或 150 nsec（短）双极脉冲，在存在 2.5 mM（低）或 20 mM（高）添加 CaCl2 的情况下，将浓缩的正常供体 PRP 激活。通过血栓弹性图 (TEG) 评估凝块形成。通过流式细胞术评估 α 颗粒（P-选择素）和 T 颗粒（TLR9 和蛋白二硫键异构酶 [PDI]）表面暴露标志物。通过 ELISA 测量激活的 PRP 上清液中的因子。通过透射电子显微镜 (TEM) 评估血小板形态。
结果
与 PEF A 和 B（4.4-8.7 分钟）相比，用凝血酶（<1 分钟）形成初始凝块的时间更短，但与凝血酶或 PEF A 相比，PEF B 形成的凝块强度（从 TEG 最大幅度得出的弹性模量）更高（p<0.05）。用 PEF A 激活的 PRP 上清液中的 EGF 水平高于其他所有条件下的上清液。相比之下，在用凝血酶、PEF A、PEF B 激活后，上清液中 PF4、PDGF 和 VEGF 的水平没有显著差异。在用低或高 CaCl2 处理时，与 PEF A 或 B 相比，TLR9 和 PDI 等 T 颗粒标志物在凝血酶后更高。通过 TEM，PEF 处理的样品中的血小板保留了一组颗粒，表明调节了颗粒释放。
结论
脉冲长度和极性可以调节，以产生与凝血酶产生的一样强或更强的治疗性血小板凝胶，并且可以根据不同伤口愈合阶段的重要生长因子来调整生长因子谱。
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