Angiogenic properties of sustained release platelet-rich plasma: Characterization in-vitro and in the ischemic hind limb of the mouse
Angiogenic properties of sustained release platelet-rich plasma: Characterization in-vitro and in the ischemic hind limb of the mouse
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DOI：
10.1016/j.jvs.2009.06.016
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发表时间：
2009-10-01
影响因子：
4.3
通讯作者：
Sakata, Ryuzo
中科院分区：
文献类型：
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作者：
Bir, Shyamal Chandra;Esaki, Jiro;Sakata, Ryuzo
工作背景：虽然单一的生长因子在诱导最佳新生血管方面存在局限性，但富血小板血浆（PRP）是多种生长因子的自体储备者。然而，关于PRP相关的新生血管形成的机制知之甚少。本研究的目的是表征PRP的血管生成和生长因子含量，并在体外确定其对内皮细胞增殖的影响。此外，本实验旨在确定不同组合物的PRP（溶液与持续释放）的有效性灌注和新生血管在小鼠模型的后肢ischemia.Methods：不同的生长因子进行了测定，通过酶联免疫吸附试验（ELISA）。在体内研究中，我们使用明胶水凝胶作为PRP中生长因子的缓释载体。我们通过切除野生型C57 BL 6小鼠的右股动脉来诱导后肢缺血。手术后，将小鼠随机分配到四个实验组;对照组（C）、100 μ L缓释形式的贫血小板血浆（PPP）、100 μ L溶液形式的PRP（PRP-sol）、100 μ L缓释形式的PRT（PRY-sr）;每种制剂通过肌内注射施用到缺血后肢。终点评价是通过激光多普勒灌注成像的血液灌注、通过抗血管性血友病因子（vWF）的血管密度和通过抗平滑肌肌动蛋白（SMA）抗体的成熟血管密度。将骨髓单个核细胞移植到野生型C57 BL 6小鼠体内，建立绿色荧光蛋白（GFP+）转基因小鼠模型，用抗CD 34抗体染色检测转基因小鼠缺血区的CD 34+细胞。体内研究表明，通过激光多普勒灌注成像（LDPI）测量，PRP的持续释放增加了缺血组织的灌注（C组、PPP组、PRP-sol组和PRP-sr组分别为57 +/- 12、56 +/- 9、72 +/- 7、98 +/- 4; P <0.05）;毛细血管密度（C组、PPP组、PRP-sol组和PRP-sr组分别为151 +/- 16、158 +/- 12、189 +/- 39、276 +/- 39，P < .05）和成熟血管密度（C组、PPP组、PRP-sol组和PRP-sr组分别为28 +/- 2、31 +/- 3、52 +/- 10、85 +/- 13，P < .05）。PRP缓释剂还能增加转基因小鼠缺血部位的CD 34+细胞（6 +/- 3 vs 18 5/mm 2，P <0.05）。结论：PRP缓释剂可能通过刺激小鼠后肢缺血的血管生成、动脉生成和血管生成来恢复血液灌注。（J Vasc Surg 2009;50：870-9.）
Background: While single growth factor has limitation to induce optimal neovascularization, platelet-rich plasma (PRP) is an autologous reserver of various growth factors. However, little is known about the mechanism of PRP-related neovascularization. The objective of this investigation was to characterize the angiogenic and growth factor content of PRP and to determine, in vitro, its effect on endothelial cell proliferation. Additionally, this experiment sought to determine the effectiveness of different compositions of PRP (solution versus sustained release) on perfusion and neovascularization in a murine model of hind limb ischemia.Methods: Different growth factors were measured by enzyme-linked immunosorbent assay (ELISA). In vivo study, we used gelatin hydrogel as a sustained release carrier for growth factors in PRP. We induced hind limb ischemia by excising right femoral artery in wild type C57BL6 mice. After surgery, mice were randomly assigned to four experimental groups; control (C), 100 mu L of sustained release form of platelet-poor plasma (PPP), 100 mu L of solution form of PRP (PRP-sol), 100 mu L of sustained release form of PRT (PRY-sr); each formulation was administered via an intramuscular injection to the ischemic hind limb. Endpoint evaluations were blood perfusion by laser Doppler perfusion image, vascular density by anti Von Willebrand factor (vWF), and mature vessel density by anti smooth muscle actin (SMA) antibody. Green fluorescent protein (GFP+) transgenic mice were generated by transplantation of bone marrow derived mononuclear cells to wild type C57BL6 mice, and finally CD34+ cell in the ischemic site of transgenic mice was detected by staining with anti-CD34 antibody.Results: In vitro study showed that PRP containing different growth factors induces endothelial cell proliferation and capillary tube formation. In vivo study demonstrated that sustained release of PRP increased perfusion of ischemic tissue as measured by laser Doppler perfusion imaging (LDPI) (57 +/- 12, 56 +/- 9, 72 +/- 7, 98 +/- 4 for groups C, PPP, PRP-sol, and PRP-sr, respectively; P < .05); capillary density (151 +/- 16, 158 +/- 12, 189 +/- 39, 276 +/- 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) and mature vessel density (28 +/- 2, 31 +/- 3, 52 +/- 10, 85 +/- 13 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05). Sustained release PRP also increases CD34+ cells in the ischemic site of transgenic mice (6 +/- 3 vs 18 5/mm(2) for groups control and PRP-sr respectively, P < .05).Conclusion: Sustained release of PRP containing potent angiogenic growth factors restores blood perfusion presumably by stimulating angiogenesis, arteriogenesis, as well as vasculogenesis in the mouse hind limb ischemia. (J Vasc Surg 2009;50:870-9.)