| Objective: The immune response of valve material determines its in vivo outcome,and the modulation of immune microenvironment to induce the polarization of macrophages to M2 is expected to promote the recellularization of decellularized valves and achieve tissue remodeling.In this study,polyethyleneimine-catechol(PEI-C)and red blood cell membranes(RBCM)layer-by-layer self-assembly modified decellularized heart valves(DHV)were constructed,the feasibility of material construction was evaluated,and the stability of the valve material,in vivo and in vitro biocompatibility and its effect on recellularization were evaluated.Methods: RBCM were were obtained by hypotonic membrane rupture from SD rat erythrocytes.PEI-C was synthesized from 3,4-dihydroxyphenylpropionic acid and polyethyleneimine under the catalysis of carbodiimide.Characterizations such as particle size and zeta potential of RBCM and PEI-C were detected,and the effect of RBCM on macrophage polarization was assessed.The PEI-C-RBCM modified DHV(DHV-PEI-CRBCM)was constructed by layer-by-layer assembly from immersion of DHV in PEI-C and RBCM solution,and the modification of RBCM on the surface of the valve was detected.The hydrophobicity and stability of DHV-PEI-C-RBCM were assessed by determining the water contact angle,mechanical properties and anti-enzymatic hydrolysis properties.Macrophage lines were used to assess the effect of valves on macrophage polarization.Human umbilical vein endothelial cells were implanted in vitro on the valve surface and valve extract to assess cytocompatibility.Different components of the blood are incubated with valves to assess hemocompatibility.In terms of in vivo experiments,the subcutaneous embedding model of SD rat and the valve duct-abdominal aortic transplantation model were constructed,and the samples were obtained 7,14 and 28 days after surgery,then in vivo properties of DHV-PEI-C-RBCM such as immune cell infiltration,degradation rate,anticalcification performance,blood compatibility and recellularization were evaluated by histological and immunofluorescent staining.Results:(1)Characterization of PEI-C and RBCM: PEI-C was positive charged with the particle size about 300 nm.C-N and N-H groups were found in infrared spectrum of PEI-C.RBCM were negatively charged with the particle size about 200 nm.C=O,P=O and P-O-C groups were found in infrared spectrum of RBCM.The existance of CD47 protein in RBCM was proved by SDS-Page coomassie brilliant blue staining.The results of immunofluorescence and ELISA showed that CD206 expression was increased and proinflammatory cytokine secretion decreased after co-culture between macrophages and RBCM,indicating that RBCM had the ability to induce the polarization of macrophages to M2 subtypes.(2)Characterization of valve materials: The scanning electron microscopy results showed that the wavy fibers on the DHV surface disappeared after PEI-C modification,and the granular coating appeared on the surface after RBCM modification.X-ray photoelectron spectroscopy showed that the peak value of P2 p orbit increased after RBCM modification,the mass percentage of phosphorus increased by scanning electron microscopy,and the percentage of phosphorus in DHV increased compared with that before modification.Di I staining showed that the fluorescence intensity increased significantly after RBCM modification,indicating that RBCM was successfully modified on the surface of DHV.The results of water contact angle showed that the hydrophilicity of the valve increased after PEI-C-RBCM modification.Fourier infrared spectroscopy showed that the valve protein composition was not changed after PEI-C and RBCM modification.Tensile experiments showed that Young’s modulus and ultimate tensile strength increased significantly after PEI-C modified DHV,and the mechanical properties of the valve were basically unchanged after RBCM modification.The mechanical properties of DHV-PEI-C group and DHV-PEI-C-RBCM group were comparable to those of glutaraldehyde crosslinked natural valves.Enzymatic degradation experiments showed that the anti-collagenase degradation performance of the DHV group was poor,and the anti-enzymatic degradation performance after PEI-C-RBCM modification was significantly improved,which was similar to that of glutaraldehyde cross-linked natural valves,indicating that PEI-C-RBCM modified decellularized valves enhanced the hydrophilicity and stability of valves.(3)In vitro biological performance evaluation: DHV-PEI-C-RBCM induced the upregulation of CD206 expression in macrophages in vitro and reduced the release of TNF-α,which indicated the ability to regulate the polarization of macrophages to M2 subtypes.CCK-8detection and confocal photography of live/dead cell staining showed that the cell compatibility of DHV-PEI-C-RBCM was good,while glutaraldehyde cross-linked natural valve was cytotoxic.DHV-PEI-C-RBCM significantly reduced adhesion and activation of platelet,plasma protein adsorption,and thrombosis,demonstrated good blood compatibility in vitro,which was similar to glutaraldehyde cross-linked natural valves.(4)In vivo biological performance evaluation: The histological staining of subcutaneous embedding and abdominal aorta graft model in SD rats showed that DHV-PEI-C-RBCM had good biocompatibility in vivo.The degradation rate of DHV-PEI-C-RBCM was slower than DHV,and there was no calcification,which was better than DHV and glutaraldehyde cross-linked natural valves.Fluorescence staining of immune cells showed that DHV-PEI-C-RBCM induced the polarization of macrophages to M2 subtype after implantation,accelerated the process of tissue remodeling and recellularization.CD31 fluorescence staining showed that DHV-PEI-C-RBCM accelerates valve reendothelialization.Conclusions: By layer-by-layer self-assembly,PEI-C and RBCM could modify DHV to construct DHV-PEI-C-RBCM,which could significantly enhance the mechanical properties and stability of DHV.DHV-PEI-C-RBCM could induce the polarization of macrophages to M2 subtype,with good cytocompatibility and improved hemocompatibility of DHV.DHVPEI-C-RBCM had well-defined biocompatibility in vivo,induced M2 subtype macrophages after implantation thereby accelerating recellularization and tissue remodeling. |
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