Experimental Study On The Effects Of Three Platelet Concentrates Of Promoting Vascularization In Perforator Flap

Objectives:The vascularizing effects of platelet-rich plasma（PRP）,platelet-rich fibrin（PRF）and concentrated growth factor（CGF）on perforator flap in rats were compared to indicate their effects on the survival rate of skin flaps.Methods:Under the guidance of previous animal experiments that PRP promoted the survival rate of flaps and increased vascular density,PRP,PRF and CGF gels were prepared.Three kinds of platelet concentrate gel were prepared,and their gross and microscopic structures were observed by gross specimen,pathological section and scanning electron microscope.The enzyme-Linked immunosorbent assay（ELISA）was used to detect the release patterns of growth factors of three gels,such as vascular endothelial growth factor（VEGF）,basic fibroblast growth factor（b FGF）,transforming growth factor-β₁（TGF-β₁）and platelet-derived growth factor-AA（PDGF-AA）were determined.Next,PRP,PRF and CGF were prepared as conditioned media with two concentrations.Proliferation experiment,scratch experiment and tubule formation experiment were conducted respectively.Cell growth curve,wound healing percentage and tubule formation were measured.The expression levels of VEGF and b FGF in HUVEC were detected by Western Blot（WB）and quantitative real-time PCR（QRT-PCR）.Finally,the rat back cross-region perforator flap model was constructed.After PBS,PRP,PRF and CGF were pretreated and the target days were reached,the blood perfusion of the flap was evaluated by infrared thermal imaging,and the effects of the three on the survival rate of the flap were evaluated by the survival experiment of the flap.The number of vessels and microvessel density in choke region were evaluated by arterial perfusion angiography and IHC staining.The expression levels of growth factors VEGF and b FGF were detected by WB and q RT-PCR to verify the cell experiment.The expression levels of apoptosis-related factors Bcl-2 and Bax were detected by q RT-PCR to further explore the mechanism of promoting tissue vascularization in vivo.Results:It turned out that PRF and CGF gels are very similar in appearance,and the texture is stronger and firmer than PRP gels.In terms of microstructure,fibrin of PRP gel showed irregular clumps and loose structure.The fibrin of PRF and CGF gels showed a three-dimensional network structure with orderly arrangement and compact structure.All three gels can continuously release a certain amount of growth factors.The total release amount of PRP was the lowest,and CGF was the highest.There was no statistical difference between CGF and PRF.All the three gels were completely degraded in simulated body fluids,but the time required was different.PRP required the shortest time for complete degradation,while CGF required the longest time.PRP,PRF,and CGF can significantly promote the proliferation,migration,and tubulogenesis of HUVEC,and can significantly up-regulate the expression levels of VEGF and b FGF in HUVEC,and this effect of vascularization is concentration-dependent.CGF and PRF were significantly better than PRP in promoting angiogenesis and up-regulating VEGF and b FGF,but there was little difference between CGF and PRF.CGF and PRF have better effects than PRP,but there is no significant difference between CGF and PRF.Platelet concentrate can not only up-regulate VEGF and b FGF expression,but also up-regulate apoptosis inhibitor Bcl-2 and down-regulate apoptosis promoter Bax.Conclusion:PRP,PRF and CGF all have good vasculogenic effects,but due to the different preparation schemes of PRP and CGF,their microstructure and biological properties are different,so PRF and CGF have better vasculogenic potential than PRP.