Development, Test And Initial Application Of Pulsatile Bioreactor In Vitro

Cardiac valvular disease is a significant cause of mortality and morbidity in human being.The most common therapeutic procedure for the cardiac valvular disease is valve replacement.There are two kinds of valvular prosthesis:mechanical valve and bioprosthetic valve.Though it has been proved that all these valves are effective and has been shown to significantly alter the course of valvular disease,they are still far from an ideal one.Mechanical valves are associated with a significant risk of thromboembolism and require life-long anticoagulation.Bioprosthetic valves do not need life-long anticoagulation,but they are far less durable and are subject to progressive tissue deterioration.The tissue engineering heart valves are constructed by repopulate heart valve scaffolds with viable cells.It may becomes a biological valvular substitute that can restore and maintain the the valvular function.Tissue engineering heart valve have good histocompatibility,long durability,no immunogenecity and no need for anticoagulation.They have the ability for growth and repair.They will be better than mechanical valves and bioprosthetic valves.With the development of tissue engineering heart valves,it is found that the endothelial cells on the TEHV leaflets have very weak adhesion and are brushed off easily from the valve leaflets once under the hemodynamic condition.It is needed to develop pulsatile flow cultural system to culture TEHV.So it may improve the TEHV's ability to avoid being brushed off..However,there are no perfect pulsatile bioreactors in the world.All of them are not mature,and need to be improved.In our study,the first is to design,develop a pulsatile bioreactor in vitro and evaluate its function:whether the system can simulate the hydrodynamic character heart valve and be used in the study of TEHV.Thenthe TEHV are created under static condition by reseeding the peripheral endothelial progenitor cells(EPCs) onto the acellular porcine aortic valves.Then TEHV are cultured in the pulsatile flow cultural system.In the end the growth of endothelial progenitor cells are observed.Partâ… .(1)The development and test of pulsatile bioreactor in vitroThe pulsatile bioreactor consisted of the dynamical part and cultural part. The dynamical part are a pulsatile air pump developed by ourselves.The cultural part consists of ventricular assisted device,valve cultural chamber and reservoir.They are connected with medical silicon rubber tubes.The ventricular assisted device is provided by our department.The valve cultural chamber and reservoir are made of the Polycarbonate(PC).There was a steal valve holder between the two part of the valve cultural chamber.The described part can be placed in standard humidified incubator at 37℃and 5%CO₂.(2)The functional evaluation of pulsatile bioreactor:The first is to evaluate its mechanical stability through a bioprosthetic valve.We observe whether it can produce the pulsatile flow and work in order or not for 5 days.And we test it three times.The opening and closing of valve leaflet should also be observed..Then we verified the ability of anti-infection by the cultural test of the LB medium onto the agar board.Also we test it three times.The third is to test the hydrodynamic function.We verified its hydrodynamic function using the bioprothesis as a experimental valve The volume and pressure must be recorded in the test..The last is the cell toxicity test in vitro of materials used to create the system.We cultured the human umbilical vein endothelial cells together with the materials of PC and medical silicon rubber to observe the variation of relative grorate by MTT test.Results (1)First,according to our design,we successfully created the pulsatile reactor.The pulastile air pump worked well.The systolic pressure is about 0-250mmHg,while the diastolic pressure is about -100-100mmHg.Its frequency is about 0-90 times/min.The volume of reservoir is about 1000ml..The cultural part can be placed into the incubator. (2)The functional evaluation of pulsatile bioreactor:Through the test of mechanical stability,we found the system can producepulsatile flow and work in stabilization for 5 days with the flow adjusted from low to high gradually and had no leakage.The bioprosthetic valve open and close freely.Using the LB medium,the system had safely run for 5 days without bacterial and mildew contamination.In the cultural test of the LB medium onto the agar board,there isn't contamination of the bacterium and mildew,which showed the anti-infect function of system.From the cellular toxicity test in vitro,materials:including PC,medical silicon rubber tube can be cultured together with the endothelial cells.The results of the relative growth demonstrated that materials created the system have little cellular toxicity to endothelial cells.From the test of the hydrodynamics,the system can produce the pulsatile flow with the volume about 0-3500 ml/min,the pressure about 0-120mmHg and frequency about 10-90 beats/min.Partâ…¡.the primary study of pulsatile bioreactor in the creation of TEHVIn this study we first created a TEHV in vitro under static condition,by seeding human peripheral endothelial progenitor cells(EPCs)onto the decellularized porcine aortic valve,and then observed the growth situation of endothelial progenitor cells in the 7th day about histology,and SEM test.The second is the result of the morphology and adhesion of endothelial progenitor cells preconditioned in the pulsatile bioreactor.Specimen was divided into three groups in random.The group with preconditioning:The pulsatile circulation was started with 100ml/min.The initial flow rate was increased by 200ml/min every day until a maximal flow of 1500ml/min for one day;the group without preconditioning:the TEHV created under the static condition was directly placed into the higher flow(1500ml/min)for 8 days without preconditioning;Group control:the TEHV only created under the static condition neither preconditioning nor the higher flow.The growth situation of the endothelial progenitor cells was observed by histology,and SEM test.Results:The first,we got the decellularized porcine aortic valve performed previous details.The decellularized procedure resulted in complete removal of the cell components,however the construction of matrix was maintained.We succeeded in creating TEHV under static condition in vitro by seeding the EPCs onto these valve leaflets.The EPCs formed a continuous monolayer on the valve surface and grew well.Then EPCs of those valve leaflets without precondition,almost had been brushed off when underwent higher flow for 8 days.On the contrary,those leaflets preconditioned in system had a lot of residual cells more than the high flow group(0.132±0.013 vs 0.648±0.036,p＜0.05).Meanwhile,the connection between the cells and matrix become closely.Conclusions:We successfully developed the pulsatile bioreactor in vitro.The pressure and volume are enough to us.It can mimick the physiological hydrodynamic condition.The system can be use precondition study of the TEHV.Using the decellularied porcine aortic valve as the scaffold,seeding the Human peripheral EPCs onto it,we created the reseeded valves in vitro under static condition.The reseeded valves were placed into pulsatile bioreactor to precondition,which had more residual cells than the group without precondition when they underwent higher flow.So the preconditioning advantageous to enhancing the adhesion of the endothelial progenitor cells and acellular porcine aortic valves.