Objectives In this study we investigate the In vitro pharmacokinetic of nanoporous CREG eluting-stent (CREGES) and evaluate the efficacy and safety of nanoporous CREGES in inhibiting neointima proliferation in porcine coronary model.
Methods (1) The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000. (2) For the absorption of the CREG protein by the nanoporous stent, the stents were totally immersed and kept vertical in solution of CREG protein in a phosphate buffer. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from to investigate the cellspecific pharmacokinetic effect of CREG protein and rapamycin. (3) The nanoporous bare metal stent (BMS), nanoporous CREGES and sirolimus-eluting stent (PARNTER) were implanted in left anterior descending coronary, left circumflex coronary and right coronary artery of forty porcines in random. And after 7, 14 and 28-day, animals were sacrificed for histomorphologic and pathologic score analysis.
Results The lysates of 293F cells transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices, the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Stents eluting the CREG protein were tested for their adsorption characteristics by radioisotope technique with 125I labelled CREG protein. The amount of CREG protein adsorbed onto the nanoporous bare metal stent was dependent on the concentration and duration of immersion in the solution. We had tested three different concentrations for 48h. Maximal CREG protein binding was therefore defined as the amount of agent bound to stent wires after 48 hours immersion in a 1.5 mg/ml solution. We implant the CREG eluting stents and 316L stainless steel stents as the control in pig model, to study the bio-security validity of prevention ISR by sliding microtome, SEM, transmission electron microscope (TEM), immunohistochemistry, tissue stain and biochemistry methods.
Conclusions The nanoporous CREGES represents a novel promising device in preventing in-stent restenosis by inhibiting the thrombosis and neointimal hyperplasia via accelerating the endothelialization of the stent surface and inhibiting the smooth muscle cell proliferation.
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