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Angiogenic properties of sustained release platelet-rich plasma: characterization in-vitro and in the ischemic hind limb of the mouse.
BACKGROUNDWhile 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.
METHODSDifferent 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 muL of sustained release form of platelet-poor plasma (PPP), 100 muL of solution form of PRP (PRP-sol), 100 muL of sustained release form of PRP (PRP-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.
RESULTSIn 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).
CONCLUSIONSustained 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.
CLINICAL RELEVANCEPRP is a natural reserver of various growth factors that can be collected autologously and is costeffective. Thus for clinical use, no special considerations concerning antibody formation and infection risk are needed.Some clinical devices to automatically prepare PRP are available at present. PRP are consistently being used clinically inthe department of orthopedics and plastic surgery (oral, maxillary facial) for a long time. On the basis of researchevidence, some publications have reported positive results in either bone or soft tissue healing. However, some researchconcludes that there is no or little benefit from PRP. This is likely due to faster degradation of growth factors in PRP sincesome authors suggest using sustained release form of PRP to deliver optimal effect of PRP. Gelatin hydrogel is also beingused clinically as a slow, sustained release of carrier for growth factors in our center recently.
Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan., , , , ,
Journal of vascular surgery 50:4 2009 Oct pg 870-879.e2
Disease Models, Animal
Enzyme-Linked Immunosorbent Assay
In Vitro Techniques
Mice, Inbred C57BL
Sensitivity and Specificity
Pub Type(s)Comparative Study