Experimental Research On Construction Of Small Caliber Vascular Graft With Porcine Small Intestinal Submucosa Hybrid Scaffold

Objective: In the field of cardiovascular surgery,ePTFE or PET is often used in clinical reconstruction of large arteries.However,when these synthetic materials used in small caliber artery(diameter less than 6 mm,such as coronary artery or peripheral arteries)reconstruction,the clinical outcome is not ideal.Bad histocompatibility,easy to form thrombosis,intimal hyperplasia or infection are causes of graft occlusion.For such small artery bypass graft,autologous artery(such as internal thoracic artery and radial artery)or vein(such as great saphenous vein),are still the most ideal vascular substitutes.However,because of their poor quality or lack of quantity,they can not be easily obtained.Artificial vascular substitutes for small-caliber arteries have been a hot and difficult topic for many years.Autologous vascular parietal cells have been used as seeding cells for artificial blood vessel cultivation or implantation.Some achievements have been made,but the shortcomings are also obvious.In addition,the procedures of autologous cell pretreatment,seeding or bioreactor culture are complex and invasive,and can make graft susceptible to infection.The growing cardiovascular problems needs off-the-shelf small-caliber artificial vascular grafts that do not require tissue harvest or cell from patients.Therefore,it is necessary to develop a new type of small-caliber artificial blood vessel to meet clinical needs.In this study,we present the successful development of a hybrid“sandwich” small caliber artificial vascular graft based on biodegradable porcine SIS,with curdlan gel and DIP mixture film served as the filler for the “sandwich”.The objective of this study was to test the biological performance in vitro,patency and remodeling of the graft in rabbit common carotid artery bypass implantation model,mechanism of regulation on endothelialization and intimal hyperplasia and clinical application prospects of the hybrid small caliber vascular graft.Methods: SIS was prepared,decellularized and sterilized according to Abraham method.SIS was coated with heparin by crosslinking agent EDC.The release rate of heparin combined with SIS in vitro was tested.Curdlan was used as carrier of DIP.Different concentrations of curdlan and DIP mixture films which served as the filler for the “sandwich” based on bilayer SIS.In vitro,the release rate of DIP,PLT adhesion test,hemolysis test,artificial material on the proliferation of vascular ECs and VSMCs and toxicity test were studied.Three types of graft tubes(2.0-mm internal diameter,20-mm length)were manufactured: bilayer SIS with 10% curdlan and 10% DIP mixture film as “sandwich” grafts(SCD),bilayer SIS with 10% curdlan film as “sandwich” grafts(SC),monolayer SIS grafts(S).Biomechanical performance of artificial blood vessels was tested.The grafts were bypass implanted into rabbit NCA separately and feeding for 2-3 months.Each group contained 16 rabbits,and 16 non-surgical rabbits served as control group.Doppler and CT monitor graft patency.After explantation,followed by gross observation,intima scanning electron microscopy,qualitative and quantitative analysis of collagen and elastic fiber,morphology,immunofluorescence and Western Blot were used to evaluate the in vivo characteristics of graft reconstruction.The mechanism of regulation and influence on endothelialization and intimal hyperplasia of the hybrid small caliber vascular graft was researched through cell cycle and signal pathway detection.Results: Observed under the optical microscope with H&E staining,mucosal side of SIS prior decellularization was smooth,muscular side was rough,residual blue nuclei were still visible.Mucosal side had little change,but no any signs of remaining nuclear material could be seen after decellularization,indicating successful decellularization through the wall of SIS.Scanning electron microscope observation showed that the structure of the muscular side became looser after decellularization,forming a three-dimensional reticular stereostructure with aperture ranging from 20 to 200 ?m.After the immobilization of heparin and SIS,heparin released(18.5 + 3.5)% in the first 24 hours in vitro,then the heparin release curve of the latter 20 days was relatively gentle and gradually released(94.3+2.4)%,indicating that heparin immobilization was relatively strong by crosslinking.The quantitative analysis of fluorescence intensity showed that after 91 days of in vitro DIP release,SIS+10%curdlan+10%DIP sandwich material released(21.8±6.2)%.It was significantly lower than that of SIS+2.5% curdlan+10%DIP(67.5±7.2)% and SIS+5%curdlan+10%DIP(49.4±9.5)%.DIP release analysis results of SCD showed that approximately about(53.39±3.52)% of DIP was released at 2mo,(91.01±4.79)% was released at 3mo.PLT adhesion tests showed that PLT adhesion degree of nonheparinized SIS was high,PLT retention index %PRI=(59.2+6.7)%,after crosslinking with heparin %PRI=(11.2+2.4)%,"sandwich" SIS+10% curdlan %PRI=(9.5+2.7)%,"sandwich" SIS+10%curdlan+10%DIP %PRI=(3.3+0.6)%.Hemolysis tests showed that the hemolysis rates of all materials were less than 5%.MTT experiment showed that the higher the DIP concentration was,the stronger the inhibitory effect on the proliferation of VSMCs.DIP concentration less than 10% sandwich material promoted the proliferation of ECs.The mechanical property tests showed that the bursting strength of SCD and SC are better than that of group S.After graft implantation,all those grafts occluded and the randomized rest adding up to half of each group rabbits were sacrificed and explanted at 2-mo.Other 8 cases of each group were fed for 3mo and explanted.Follow up of Doppler show that all 8 SCD grafts were patent at 2-mo,1 occluded at 3-mo.6 of 16(37.5%)SC grafts and 5 of 16(31.25%)S grafts occluded at 2-mo.Histological analysis and immunofluorescence technique of explanted grafts demonstrated that at 2-mo,all patent grafts showed significant infiltration of host cells and varying degrees of EC coverage.The occlusion of the SC and S grafts was due to thrombosis.At 3-mo,1 SCD occluded,patent SCD grafts showed a confluent and functional endothelium,no intimal hyperplasia and the neointima layer comprised of circumferentially aligned VSMCs.SC grafts and S grafts showed incomplete endothelialization and varying degrees of mural thrombus,accompanied by the performance of SC graft occlusion(3 of 8),S graft occlusion(2 of 8).Qualitative and quantitative analysis of collagen from SCD-3mo showed that the proportion of collagen was close to that of rabbit NCA.The proportion of elastin in each group was lower than that in NCA,and there was no evidence of fibroelastin formation,indicating that tissue remodeling was imcomplete.Western Blot analysis showed that the expression of eNOS in SCD was higher than that in SC and S,and the expression of ?-SMA and MYH was lower than that of SC and S,indicating that DIP has the effect of promoting endothelialization and inhibiting the proliferation of VSMCs.Cell cycle detection showed that DIP could increase the proportion of G2 and S phase in ECs.Hcy inhibited EC proliferation in G1 phase,and DIP partially reversed the effect of Hcy on EC cell cycle.On the contrary,Hcy can increase the proportion of G2 and S in VSMCs.DIP can make VSMC proliferation stagnate in G1 phase.DIP can partially reverse the effect of Hcy on the cell cycle of VSMCs.Interventions of Hcy,DIP and different blockers may lead to changes in cAMP dependent signaling pathways which affecting the function of HUVECs.Interventions of Hcy,DIP and different blockers may lead to changes in cAMP-dependent signaling pathways which affecting the proliferation of HASMCs.Conclusion: In vitro studies showed that fresh SIS achieved heparinization and sustained release with the aid of EDC cross-linking after a series of decellularization and disinfection procedures.The hybrid small caliber vascular graft with 10%curdlan+10%DIP mixed membrane as sandwich of bilayer SIS had good biomechanical properties,blood compatibility and ECs compatibility,and can inhibit the proliferation of VSMCs.With curdlan as carrier,DIP can achieve long-term sustained release.The results of animal experiments showed that cross linked heparin and curdlan loaded DIP drug delivery system as sandwich of artificial vascular graft enhanced the adhesion ability of PLT and effectively prevented thrombosis.DIP inhibited the excessive proliferation of artificial vascular intima and promoted early endothelialization.Signal pathway studies showed that DIP can reverse Hcy-induced HUVECs dysfunction through cAMP-dependent PKA-RhoA and Epac-PI3 K pathways.DIP can inhibit the proliferation of HASMCs induced by Hcy through cAMP-dependent PKA and Epac-MEK pathways.