Study On The Construction Of Tissue-engineered Anti-calcification Heart Valve By Modifying Decellularized Porcine Aortic Valve Scaffold With Mesoporous Silica Loaded With SiRNA

Objective:Heart valve disease is a serious dynamic and progressive cardio-macrovascular surgery disease,and valve replacement is currently the only clinically effective treatment that can change the outcome.However,postoperative calcification and embolism leading to valve failure have always been problems that medical staff need to overcome.We constructed a tissue engineered heart valve with decellularized porcine aortic valve to explore an ideal replacement valve based on its good histocompatibility,self-repair and lifetime durability.In this way,the two major serious complications of pathological calcification and platelet thrombosis in patients with valvular heart disease can be improved,and the risk of needing a second operation can be reduced,and the double burden of economy and psychology of patients can be reduced.Methods:1.Rat VICs were extracted by digestion method,and were observed microscopically,and were further confirmed by α-SAM and Vimentin staining.2.The calcification model of VICs in vitro was constructed,and the transcription,translation,Alizarin red staining and alkaline phosphatase staining of VICs were detected to evaluate the model,and siRNA-Runx2 with the highest transfection efficiency was screened out.3.The nanoparticle was used to further modify the load,and finally the nanoparticle drug carrier MSN@VEGF-PEI-siRNA was prepared.The characterization and cytotoxicity of the nanoparticle drug carrier were examined.4.Nanoparticle drug carrier MSN@VEGF-PEI-siRNA was used for transfection of VICs,and the anti-calcification effect of the nanoparticle drug carrier was evaluated by detecting the transcription and translation of valve interstitial cells.5.Nanoparticle drug carrier MSN@VEGF-PEI-siRNA was added to co-culture with rat endothelial cells,and the ability of nanoparticle drug carrier to accelerate endothelialization in vitro was evaluated through a series of physiological activities of endothelial cells6.The new composite valve scaffold was prepared by loading MSN@VEGFPEI-siRNA on decellularized porcine aortic valve with heparinization,and its biological properties were characterized and evaluated by scanning electron microscopy.Results:1.Microscopic observation showed that the VICs were fusiform and irregular,and immunofluorescence staining showed positive antibodies to α-SAM and Vimentin.2.The results of m RNA expression and protein translation showed that the calcification model of VICs in vitro was established successfully,and the siRNA silencing gene RunX2 was used,and the results of PCR,Western blot and staining showed good anti-calcification performance.3.SEM and TEM were used to characterize MSN,MSN@VEGF-PEI-siRNA.The results showed that the particle size of the three groups was about 49 nm,and the suitable concentration(0.75mg/m L)showed no cytotoxicity,and the drug loads were82.37%(VEGF)and 78.81%(siRNA),respectively,with good dispersion.4.The results of m RNA expression,protein translation and staining showed that MSN@VEGF-PEI-siRNA had a good transfection efficiency.5.MSN@VEGF-PEI-siRNA nanocarriers showed better effects than MSN and MSN-PEI-siRNA in the proliferation,adhesion,tubulation and migration of rat endothelial cells.6.Scanning electron microscopy showed that the new functionalized valve scaffold DPAV-MSN@VEGF-PEI-siRNA was successfully constructed,and initially showed the effect of anti-calcification and accelerating endothelialization.Conclusion:The new composite valve scaffold based on mesoporous silica nanoparticles loaded with siRNA-RunX2 and VEGF modified decellularized porcine aortic valve shows ideal anti-calcification and accelerated endothelialization capabilities,and is expected to become a new type of valve replacement.