Construction Of Covalently Modified Decellularized Porcine Aortic Heart Valve With Osteoprotegerin In Vitro

Background:With the aging of society,heart valve disease has become a public health problem that threatens human health,and the morbidity has increased year by year.Among them,calcific aortic valve disease(CAVD)occupies a very important position in heart valve disease.At present,there are no effective measures and drugs to prevent or treat CAVD.Heart valve replacement is the most effective method for the treatment of CAVD.Mechanical valves and biological valves are two main types of prosthetic heart valves,but they have certain defects.Patients may have bleeding and thrombotic complications after implantation of mechanical valves and require anticoagulant medication for life after surgery.Although biological valves have good biocompatibility,but they are prone to calcification and degeneration,and patients may face the possibility of reoperation after 10-15 years.Therefore,it has always been the goal of a cardiac surgeon to develop an ideal valve that is comparable to a normal valve.Objective:In this experiment,osteoprotegerin(OPG)was covalently modified by Michael addition reaction to decellularized porcine aortic valve,and the optimal reaction conditions for this reaction were explored,and it provided new ideas for the modification of tissue engineered heart valve materials.Methods:First,the thiol group was introduced into the decellularized aortic valve by using N-succinyl-S-acetyl nonyl glycolate(SATA),the 4-arm PEG-acrylate was attached to the collagen of the decellularized valve via Michael addition reaction,and then the cysteine residue of osteoprotegerin(OPG)was covalently bound to the acrylate in 4-arm-PEG-acrylate which cross-linked on the decellularized valve,we explored optimum binding conditions under different concentrations of OPG solution and different reaction time points.Hematoxylin-eosin(HE),Van-Greson(VG)staining,scanning electron-microscopy and immunofluorescence microscopy were performed to determine the decellularization and PEGylation effect.Cytotoxicity and mechanic properties of the engineered valves were tested.Results:1.HE staining: The normal porcine aortic valve(PAV)contained a large number of cells with intact structure,while no residual nucleus was detected in decellularized porcine aortic valve(DPAV),suggesting that the cells were effectively removed.The extracellular matrix in DPAV remained intact and arranged like wave.PEG-DPAV was morphologically similar to DPAV under microscope.2.VG staining: Collagen fibers were stained red and arranged like wave in the normal valve with black nuclei,while in DPAV and PEG-DPAV no remnants of the nuclei were observed and the fibers remained intact.3.SEM: The surface of normal valve was covered by cells without apparently naked fiber.No residual cells were observed on the surface of DPAV and PEG-DPAV.However,the fiber structure was more compact in PEG-DPAV than DPAV.4.Optimization of DPAV modifications with OPG: Based on coupling efficiency and cost,it was determined that the optimal reaction conditions were to react a piece of the valve leaflet(1 cm × 1 cm in size)in 1 ml of 500ng/ml OPG for 2 hours.5.Immunofluorescence microscopy: Under the fluorescence microscope,there were large amount of fluorescence signals in OPG-PEG-DPAV,while on fluorescence was observed in PEG-DPAV,this confirmed that OPG was successfully linked to the DPAV.6.Cytotoxicity test: The OPG-PEG-DPAV was nontoxic,the cell proliferation rate was(103.05±3.24)% and(106.83±6.17)% at 24 and 48 hours,respectively.7.Mechanical properties: The radial and circumferential mechanical properties of OPG-PEG-DPAV were comparable to normal valves.Conclusion:1.The histomorphological results showed that the decellularization effect was successful,the cells were effectively removed,the structure of valve fibers was complete,and the fiber structure after PEG cross-linking was more compact.2.Our work demonstrated that we had successfully prepared OPG-modified decellularized valve and obtained optimum binding condtions.The OPG-PEG-DPAV had no cytotoxicity,the mechanical properties were comparable to normal valves.3.This study provided a basis for modification of tissue engineering heart valve scaffold materials in vitro and provided a new idea for the construction of tissue engineered anti-calcification heart valves.