Effects Of Extracorporal Shock Wave Therapy On The Treatment Of Ischemic Skin Flap With Endothelial Progenitor Cells And Associated Mechanism Research

Background: EPCs transplantation is an effective way to treat ischemic disease, butits protective effect on ischemic tissue is limited. Low survival of the transplantedEPCs after implanted into the ischemic tissues may be responsible for the decreasedthe effect of the cell transplantation. ESWT is a promising strategy for angiogenesis.We hypothesized that ESWT may substantially increase the effect of EPCstransplantation and improve the survival of the ischemic skin flap when EPCstransplantation is combined with ESWT.Objective: To study the effect of ESWT on the transplantation of EPCs to the ischemicskin flap, and explore the mechanism of the combined strategy in the treatment ofischemic skin flap.Methods: EPCs were obtained by ex vivo cultivation and were divided into20subgroups according to the shock wave energy flux densities and shocks. ESWT wasapplied on the EPCs according to the division of the groups. After the application ofESWT, the proliferation of EPCs, the expressions of the cytokines, the tube formationability of EPCs were examined to study the effect of ESWT on EPCs, and the mostoptimized parameter for ESWT was also determined. Sixty-four SD rats were dividedin4groups: group1(Sham group), group2(EPCs transplantation), group3(ESWTgroup), group4(ESWT+EPCs). Random skin flaps were made on the backs of rats.The middle portions of the skin flaps were the place where autologous EPCs weretransplanted and ESWT were applied. The survival of the skin flaps was observedperiodically. Moreover, two more study groups were set up to clarify the exact objectof the ESWT: ESWT pretreated skin flap group and ESWT pretreated EPCs group. Thesame animal model was adopted and the survivals of the skin flap among differentgroups were compared.Results: EPCs with typical morphology could be obtained by ex vivo cultivation andidentified by fluorescence activated cell sorter and cell fluorescence staining. WhenEPCs were treated by low energy shock wave(0.04~0.13mJ/mm2), the expressions ofeNOS, Ang-1, Ang-2and BCL-2increased and the expression of IL-6, FGF-2, CXCR4, VEGF, BAX, Caspase-3decreased. SDF-1changed without statistic significance. Whencells were treated by high energy (0.16mJ/mm2) shock wave, most of theexpressions of cytokines declined except the apoptotic factors and FGF-2, and cellstended to apoptosis. The proliferation activity and the ratio of Ang-1/Ang-2reachedtheir peak values when cells were treated by ESWT with the intensity ranging from0.10mJ/mm2to0.13mJ/mm2and shocks number ranging from200to300impulses.Meanwhile a minimal value of the ratio of BAX/BCL-2was observed. In in-vivoexperiment, the survival of the skin flap in combined group was87.5±10.23%, muchhigher than other groups. In the combined treatment group, histological examinationshowed higher capillary density and a large number of vWF+cells could be observed.The expressions of chemotactic factors and angiogenic factors were higher. A similareffect could be obtained in the ESWT pretreated skin flap group. However, the effectin ESWT pretreated EPCs group was similar to the untreated EPCs group.Conclusion: ESWT could improve the survival of free skin flap treated by EPCstransplantation. The possible mechanism may be decreasing the apoptosis,increasing the expressions of angiogenic factors and chemotactic factors, which mayprovide an advantageous microenvironment for the EPCs to reside and exert theangiogenic activity effectively, thus improves the survival of the ischemic tissue.