Decellularized Porcine Pericardium With Double Network Hydrogel Coating To Improve The Biological Properties Of Bioprosthetic Heart Valves

Background:In recent years,the usage of bioprosthetic heart valves(BHVs)in patients with valvular heart disease(VHD)has rapidly increased due to the aging population and the popularity of transcatheter aortic valve implantation(TAVI)technology.The clinically used BHVs are crosslinked with glutaraldehyde(Glut).However,BHVs derived from Glut-treated porcine pericardium fail to sustain the expected durability as a result of disadvantages such as valve thrombus,cytotoxicity,endothelialization difficulty,immune response,and calcification.Multiple optimized strategies based on the disadvantages above were designed to modify the properties of Glut-treated BHVs.In this study,the porcine pericardium tissue was decellularized to eliminate xenogeneic antigens,and subsequently coated polyacrylamide/hyaluronic acid(P/H)double network hydrogel.This double network hydrogel has an interpenetrating network of special structure combining the advantages of both high-strength and biocompatible components,and several biological properties can be obtained.In the decellularized porcine pericardium with double network hydrogel coating,the hyaluronic acid hydrogel cross-linked by ionic bonds has the effects of promoting endothelialization,anti coagulation anti-inflammation and anti-calcification;The hydrophilic polyacrylamide hydrogel cross-linked by covalent bonds is able to facilitate the anti-fouling and anti-coagulation properties of the materials,and stabilize the integrity of extracellular matrix.The two intersected networks disperse the stress on the material to enhance its biomechanical properties,and ultimately improve its durability.The purpose of our study is to confirm whether combining decellularization with the synergistic effect of double network hydrogel improves these above-mentioned biological properties of BHVs.Methods:In in-vitro tests,attenuated total reflection fourier transform infrared spectroscopy(ATR-FTIR),scanning electron microscope(SEM),energy dispersive spectroscopy(EDS),ematoxylin-eosin(HE)staining,Masson staining and immunofluorescence staining were utilized to confirm the morphologies and chemical characterization of decellularized glutaraldehyde-treated porcine pericardium(dCell-PP),hyaluronic acid hydrogel-coated dCell-PP(HA-PP),polyacrylamide hydrogel-coated dCell-PP(PAAmPP)and PAAm/HA double network hydrogel-coated dCell-PP(P/H-PP).The tests of water contact angle,protein adsorption,platelet adhesion,hemolysis and arteriovenous shunt(AV-shunt)assay were applied to assess hemocompatibility.Endothelialization was observed by human umbilical vein endothelial cells(HUVECs)viability assay,HUVECs proliferation assay and HUVECs migration assay.Inflammation was observed by cell viability assay of RAW 264.7 Cells,enzyme linked immunosorbent assay(ELISA)assay and immunofluorescence staining.After accelerated calcification treatment,EDS mapping and alizarin red staining were utilized to evaluate the ability of anti-calcification.Collagenase,elastase,glycosaminoglycans degrading enzymes(GAGase)degradation tests were were used to observe component stability.Biomechanical properties were assessed by uniaxial tensile test,anti-suture tearing test,pulsatile flow test and accelerated fatigue test.After implanted subcutaneously in rats for 30 days,materials stability and biocompatibility in vivo were evaluated by using of histological analysis,immunohistochemistry staining,immunofluorescence,Masson staining,victoria blue staining,alcian blue staining,alizarin red staining and Von kossa staining.Results:In vitro,it was confirmed that the decellularization process was successful and the hydrogel existing outside the porcine pericardium did conform to the chemical composition and structure of HA and PAAm.In terms of hemocompatibility,we found that P/H-PP adsorbed less protein than Glut-PP and had an effective anti-platelet adhesion ability than Glut-PP.After circulation of rabbit AV-shunt assay for 2 h,the section and surface images showed that the lumen of P/H-PP was almost clean,with a few small thrombi on the surface,while the lumen of the dCell-PP and Glut-PP was completely blocked and numerous thrombus formed on the surface.In the aspect of endothelialization,dCell-PP without hydrogel coating was unable to support the growth of HUVECs,and on the contrary,P/H-PP could facilitate the adhesion,proliferation and migration of HUVECs.As to inflammation responses,P/H-PP did not trigger inflammation compared with other groups.In the aspect of calcification,numerous nodules of calcium deposits were observed in the Glut-PP,dCell-PP and HA-PP,but no visible calcification was detected in the PAAm-PP and P/H-PP.To assess the stability of extracellular matrix(ECM),collagenase,elastase and glycosaminoglycans degradation enzyme were used to treat the specimens.P/H-PP,PAAm-PP,and Glut-PP showed significantly lower weightloss ratios compared with that of dCell-PP and HA-PP.We further evaluated the biomechanical properties,the ultimate tensile strength(UTS)and Young’s modulus of P/H-PP were significantly higher than that of dCell-PP.Under different cardiac outputs,heart rate and mean arterial pressure,effective orifice area(EOA)and total regurgitant fraction of P/H-PP all conformed the requirements of ISO 5480.As to the durability,after 150 million cycles,EOA and total regurgitant fraction of P/H-PP still met the requirements of ISO 5480.In vivo,after implanted subcutaneously in rats for 30 days,P/H-PP showed better ECM stability,mitigated inflammation response,and reduced calcification than Glut-PP and dCell-PP.Conclusion:All these results indicate that we develop a novel approach based on decellularized porcine pericardium with double network hydrogel coating to improve the biological properties of BHVs,such as the effects of extending durability,accelerating endothelialization,anti-coagulation,anti-inflammation and anti-calcification.In the future,this strategy has the potential for creating a variety of double network hydrogelcoated hybrid biomaterials.