Study On The Mechanism Of Improving The Comprehensive Properties And Anti-calcification Of Bovine Jugular Vein Cross-linked By Glutaraldehyde By Tannic Acid-ferric Chloride Combined Treatmen

BackgroundThe glutaraldehyde(GA)fixed bovine jugular vein conduit(BJVC)was wildly applied in the reconstruction of the right ventricular outflow tract(RVOT)in patients with congenital heart disease(CHD),but the elastin component in the extracellular matrix of BJVC can easily degrade after implantation and lead to calcification,affecting its durability.Previous studies have found that tannic acid(TA)can stabilize elastin and TA treated materials achieved significant anti-calcification effects after subcutaneous implantation in rats.However,TA is prone to self-polymerization,which leads to excessive hydrogen bond cross-linking with tissue protein,reducing tissue flexibility,and affecting its clinical use.Metal ions can coordinate with TA,and trivalent iron ions(Fe3+)have a strong affinity for TA.We assume that the combined treatment of TA-ferric chloride(FeCl3)may introduce Fe3+to occupy the active site of TA and reduce its selfpolymerization,thus improving the flexibility of the treated BJVC.Moreover,the effect on other properties of the material,such as anti-calcification property,mechanical properties and the biocompatibility of the treated material is also very important to be explored.Therefore,the purpose of this study is:(1)to explore the feasibility and optimal TA-FeCl3 treatment scheme to improve material flexibility;(2)to evaluate the anticalcification,mechanical properties,and biocompatibility of BJVC treated with TAFeCl3.MethodsThe fresh BJVCs were fixed with 0.6%GA solution according to the Contegra(?)treatment protocol,and then treated with 0.3%TA solution or a combination of TA and FeCl3 solution,respectively.The combined treatments were divided into different groups according to the addition order of reagent,temperature and pH.Macroscopic observation and two-point bending test were used to evaluate the BJVCs flexibility.After 21 days or 60 days of subcutaneous implantation,the calcium ion content of the BJVCs was determined by inductively coupled plasma emission spectrometry to evaluate the anticalcification performance of the material.Then paraffin sections were prepared,and von Kossa(VK)staining was used to locate calcium deposition sites in tissues,and elastic fiber staining(Elastin Van Gieson staining,EVG)and Masson staining were used to evaluate the extracellular matrix integrity.Uniaxial tensile experiment was performed to determine the biomechanical parameters such as axial and circumferential tensile strength,elongation at break and elastic modulus to evaluate the biomechanical properties.Based on the combination of anti-calcification effect,compliance and mechanical properties,an optimized TA-FeCl3 treatment method was determined,then the biocompatibility of the treated BJVCs was evaluated in accordance with GB/T 16886,including cytotoxicity test,hemolysis test,in vitro dynamic coagulation test,D-dimer activation test and complement activation.The BJVC samples treated with GA and TA were used as control.Results(1)Macroscopic observation and two-point bending test results showed that GAfixed BJVC treated with Fe3+before TA treatment under room temperature and alkaline conditions(pH=8)had significantly improved compliance compared with TA group;(2)The anti-calcification performance of GA-fixed BJVC treated with Fe3+before TA treatment at room temperature was significantly improved after subcutaneous implantation,as the calcium content was lower than that of GA group and TA group after 60 days of implantation,and the increase of calcium content after 21 to 60 days was lower than that of TA group;(3)Von Kossa staining showed that the calcification sites of elastic fibers in TA-FeCl3 groups were significantly reduced,while GA group were densely distributed along the elastic fiber,which was consistent with the quantitative results of calcium content;(4)EVG staining and Masson staining showed that the elastic fibers of the GA group were obviously degraded,the collagen fiber structure was loose,while the extracellular matrix of TA-FeCl3 group had intact structure,unchanged morphology and good structural integrity;(5)Uniaxial tensile tests showed that the biomechanical parameters such as tensile strength,elongation at break and elastic modulus of TA-FeCl3 treated materials were not inferior to those of GA groups,indicating that the anti-calcification treatment scheme did not affect the biomechanical properties of the materials;(6)Biocompatibility tests results indicated that the BJVCs treated by the optimized TA-FeCl3 method was non-cytotoxic,non-hemolytic,and did not activate endogenous coagulation system and complement system,thus met the biosafety requirements of the GB/T 16886 for implanted medical devices in long-term contact with blood.ConclusionGA-fixed BJVCs treated by TA-FeCl3 under room temperature and alkaline conditions(pH=8)achieved improved vascular compliance,enhanced anti-calcification effect of subcutaneous implantation in rats,good structural integrity of extracellular matrix,good biomechanical properties and biocompatibility.The BJVCs modification scheme is expected to further improve the comprehensive effect of GA-fixed BJVCs in reconstruction of right ventricular outflow tract in patients with congenital heart disease.BackgroundTo understand the mechanism of BJVC xenografts calcification is of great significance for developing anti-calcification strategies.Calcification in biological xenografts is an active process involving multiple biochemical and molecular mechanisms.Previous studies have demonstrated that inflammatory and matrix metalloproteinases(MMPs)are directly related to calcification in bioprosthetic valves;Osteogenic and cartilaginous structures are also found in severely calcified areas in some xenografts.It is known that the osteogenic transformation of vascular wall cells is involved in the calcification process of senescent vasculars,and the osteogenic differentiation of vascular smooth muscle cells(VSMCs)induced by Runt-related transcription factor 2(Runx2)activation is an important pathway of vascular calcification.However,its role in xenograft calcification has not been clearly reported.Therefore,this study aims to:(1)investigate whether inflammatory,MMPs and Runx2 induced VSMCs osteogenic differentiation are involved in BJVCs xenograft Calcification and(2)investigate the possible protective mechanism of TA-FeCl3 combined treatment.MethodsBJVC valved conduits treated with GA(GA group)or TA-FeCl3(Fe-TA group)were implanted into the RVOT of sheep(n=3 for each group).The conduits were explanted 3 months(n=1)or 6 months(n=2)after surgery.The observation items include:(1)the surgical operability of valved conduits;(2)the functional status of valved conduits after anastomosis monitored by monthly echocardiography after surgery;(3)macroscopic observations were conducted on the explants and sheep organs at the endpoint of animal experiments;(4)the explants were embedded in paraffin.HE staining was conducted for histological analysis,and EVG staining for extracellular matrix analysis,and VK staining for locating calcium deposition;(5)inductively coupled plasma atomic emission spectrometry was used to quantitative analysis of calcium content in grafts;(6)Immunohistochemistry was conducted to detect markers expression of macrophages(CD68,CD163),T cells(CD3),MMP(MMP-2,MMP-9)and VSMCs osteogenic transformation(Runx2).Results(1)Surgical performance:The two groups’ conduits were of well operability,and the echocardiography after anastomosis showed that the valves worked well without blood reflux;all animals were in good health after surgery and reached the endpoint of the experiments;(2)Monthly echocardiography results showed that the grafts functioned well in the animals without stenosis or reflux of the valve;(3)Macroscopic observation:The main organs of the animal function well without lesions;and the Fe-TA group conduits inner wall was smooth without thrombosis or vegetative formation,and the leaflets were soft without adhesion or deformation;however,there were thrombosis and neointimal formation in the GA group,and the leaflets thickened and adhered to the wall,limiting its activities;(4)Calcium quantification:In the Fe-TA group,the calcium content after 180 days of implantation was comparable to that of the non implanted background,while the GA group conduits were severely calcified;(5)Histology:HE staining showed that cells only infiltrated into the adventitia in Fe-TA group while there were a large number of inflammatory cells infiltrating into the vascular media and intimal hyperplasia in the GA group;VK staining showed that only several calcification points in the Fe-TA group were located in the medial layer with abundant elastic fibers,and a large area of calcification occurred in the intimal hyperplasia area of the GA group;(6)Immunohistochemistry:Runx2 positive staining was detected in calcified areas in both Fe-TA and GA groups with α-SMA co-positioning;and the expression of CD3,MMP-2 and MMP-9 in Fe-TA group significantly decreased compared with GA group.ConclusionThe Fe-TA group conduits were of well operability,and the 180 days implantation in sheep further demonstrated the excellent anti-calcification performance of the Fe3+-TA treatment.The mechanism may be related to reducing intimal hyperplasia formation and reducing the secretion of MMP-2 and MMP-9.In addition,this study firstly reported the expression of osteogenic marker(Runx2)in SMCs-like cells in the calcified region of BJVC xenograft,which may be important to the progression of anti-calcification strategies of GA-fixed BJVCs.