The Study Of Dihydroxy-iron/heparin-VEGF Nanoparticles Modification Improves Biocompatibility And Endothelialization Of Decellular Vascular Matrix

Part I Preparation of dihydroxy-iron/heparin complexes nanomodified decellular vascular matrix and evaluation of its biocompatibilitiesObjective:There are risks of thrombosis after decellular xenograft implantation, the purpose of this study was to improve the biocompatibilities of decellular vascular matrix by dihydroxy-iron/heparin complexes (DHCs) nanomodification and evaluate the possibility of method which as a novel heparin modification for decellular xenograft.Methods:A novel thrombo-resistant surface for decellular xenograft had been developed by alternating linkage of dihydroxy-iron and heparin to decellular bovine jugular vein (DC-BJV) using dihydroxy-iron (DHI) as crosslinker. Toluidine blue colorimetric method was used to measure residual content of heparin solution after each assembly cycle, and the amount of linked heparin was calculated. Surface characterization of dihydroxy-iron/heparin layer-by-layer-assembly modified BJV (LBL-BJV) was assayed using scanning electron microscopy (SEM) and toluidine blue staining. Its biomechanical stability was detected by tensile test. The binding force of DHCs on surface of BJV was evaluated by shaken-wash test. And its biocompatibilities were detected using antithrombogenicity, platelet adhesion and cytotoxicity assay.Results:Toluidine blue colorimetric method showed the amount of linked heparin was about808±86μg/cm-1per assembly-cycle. SEM images proved DHCs were uniformly linked to and formed nanoscale films around the fibrils of DC-BJV. Toluidine blue staining histology pictures showed DHCs were mainly linked to DC-BJV surface. Washing test proved DHCs were firmly linked to BJV and sustainedly released heparin for a long time. Tensile test showed that biomechanical stability was increased. Hemocompatibility evaluations showed that PT and APTT of all trial groups were above the normal reference ranges, and mean platelet count per10000μm2area was8±4for LBL-BJV vs.48±16for DC-BJV. The endothelial cells (ECs) proliferation test showed the number of ECs on luminal surface of LBL-BJV was very similar to DC-BJV at7-day incubation. Calcium content assay showed the mean calcium content was8.5±1.9μg/mg dry weight for LBL-BJV vs.26.6±3.7μg/mg dry weight for DC-BJV at30days and21.5±6.8μg/mg dry weight for LBL-BJV vs.112.6±16.9μg/mg dry weight for DC-BJVs at60days, respectively.Conclusions:DHCs nanomodification improves biomechanical stability and biocompatibilities of decellular xenograft. Part Ⅱ Preparation and in vitro evaluation of dihydroxy-iron/low-molecular-weight-heparin nanoparticleObjective:The half-life of low molecular weight heparin (LMWH) is short, and its antithromboticity doesn’t maintain for a long time. The purpose of current study was to prepare thrombo-resistant DHI/LMWH nanoparticles (DLN) which sustainedly released LMWH and evaluate the possibility of which as a novel anticoagulatant drug.Methods:A novel thrombo-resistant DLN was prepared by LMWH and DHI under ultrasonic oscillation or magnetic stirring. The shape, size, zeta potential and encapsulation efficiency of DLNs were detected, and the difference of DLN and LMWH were analyzed by infrared absorption spectrum.Results:Average size of DLN was less than100nm under ultrasonic oscillation, and average size of dihydroxy-iron/low-molecular-weight particle was more than100nm under magenic stirring. But ultrasonic osscillation and magenic stirring did not affect the zeta potential and encapsulation efficiency of nanoparticles, their values of zeta potential were negative value, and their encapsulation efficiency was positively correlated to weight-ratio of DHI/LMWH. The average size of DLN was less than100nm when the weight-ratio of DHI/LMWH was less than1.2, and their average size was more than100nm and formed micro-particles when the weight-ratio of DHI/LMWH was more than1.2. SEM immages showed the shape of DLN was rectangle. Compared infrared absorption spectrums of DLN and LMWH, there were1732,1549,690and423cm-absorbtion peak appeared in infrared absorption spectrum of DLNs. DLNs were sustainedly released LMWH in PBS for a long time.Conclusions:DLN can be prepared via LMWH and DHI under ultrasonic oscillation, and it is a novel thrombo-resistant nanoparticle. Part Ⅲ The study of dihydroxy-iron/heparin-VEGF nanoparticles modification improves biocompatibilities and endothelialization of decellular vascular matrixObjective:Poor biocompatibility and lack of endothelialization are limitations of decellular xenograft, the purpose of current study was to improve the biocompatibilities and endothelialization of decellular vascular matrix via DHI/LMWH-VEGF nanoparticle modification, and evaluate the possibility of method which as a novel modification for decellular xenograft.Methods:A DHI/LMWH-VEGF nanoparticle modified BJV (DLN-VEGF-BJV) was developed as follows:DC-BJV was preteated by DHI and LMWH, then DHI/LMWH nanoparticles (DLNs) were linked to sueface of DC-BJV via DHI and LMWH, and then VEGF was immobilized to surface of DLN via LMWH. Its surface characterization was assayed using SEM and toluidine blue staining; its biomechanical stability was detected by tensile test; the binding force of DLNs on surface of BJV was evaluated by shaken-wash test; its biocompatibilities were detected using platelet adhesion, cytotoxicity and anti-calcification assay; and its stumilating-endothelial-cell-growth was evaluated by endothelial cells proliferation assay.Results:SEM images proved DLNs were uniformly linked to the fibrils of BJV. Toluidine blue staining histology pictures showed DLN were mainly linked to BJV surface. Shaken-washing test proved DLN-VEGF-BJV sustainedly released LMWH and VEGF for a long time. Tensile test showed that biomechanical stability was increased. Static platelet adhesion assay results showed that the modified BJV drastically decreased platelet adhesion, and the mean platelet count per10000urn2area was12±4for DLN-VEGF-BJV vs.48±for DC-BJV. The Endothelial cells proliferation in vitro assay showed ECs could adhere and proliferate on luminal surface of both DLN-VEGF-BJV and DC-BJV, and the number of ECs on the luminal surface of DLN-VEGF-BJV was more than that on DC-BJV at3-day and7-day incubation, respectively. Calcium content assay showed the mean calcium content was8.1±1.7μg/mg dry weight for DLN-VEGF-BJV vs.26.6±3.7μg/mg dry weight for DC-BJV at30days and23.5±6.1μg/mg dry weight for DLN-VEGF-BJV vs.112.6±16.9μg/mg dry weight for DC-BJVs at60days, respectively. Conclusions:The DHI/LMWH-VEGF nanoparticles modification improves biomechanical stability, antithrombogenicity, anticalcification efficacy and endothelialization of decellular vascular matrix.