In Vivo Study Of MAPCs/EPCs Tissue Engineering Venous Valve

Background and significance:It may be critical to establish tissue-engineering bioprosthetic venous valves to treat chronic venous insufficiency. However, there are no satisfying tissue-engineering bioprosthetic venous valves for implying. To develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency, a manufactured, tissue-engineered, nonimunogenic, nonanticoagulant venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of chronic venous insufficiency.Until now, few reports were found on this field and many challenges and questions remained to be solved. So, it is very significant to carry out this study of tissue-engineered venous valves. Pavcnik et al. used the mental rack with SIS as grafts of venous valves, and replanted the grafts to external jugular veins of sheeps. After one month, autogeneic EC and fibroblasts can immigrate into the grafts, so the grafts performed the functions of venous valves. But some grafts were accompanied severe complications of bleeding, thrombus, and calcification, because of without reendothelialization. Teebken et al. applied the theories of tissue engineering, used the allochthonous decellularized venous valves as scaffolds, and seeded myofibroblasts and EC to construct tissue engineered venous valves. But myofibroblasts can not grow into the walls of scaffolds. After being replanted, the grafts can not perform the long term function.In 2008, in our Institute of Biomedical Engineering, Dr. Wen Yu carried out a study of "Canine MAPCs/EPCs Tissue Engineering Venous Valves applications basis" under the guidance of the Professor. Zhang. Her works were to implant seeding cells MAPC and EPC which were derived of the receptor canine bone marrow to the allogenic acellular venous scaffold material and construct tissue engineering venous valves in vitro. Then she transplanted TEVV into the external jugular vein of recipient canine and observed effective function for 3-month after operation in vivo. The results showed the canine TEVV could play it's physiological functions after 3 months in the external jugular vein, but compared to the normal venous valve function, there was a certain degree of weakening. The reasons may be due to the seeding cells EPC which were implanted only along the direction of blood flow and training, but not against the direction of blood flow so that the back of valve could not be reendothelialized. In addition, her research was to implanted TEVV into canine's external jugular vein, blood flow are different between external jugular vein and low limb vein. Besides, 3 months for effectiveness research in vivo do not meet international standard which were accept for tissue engineering products pre-clinical effective studies in vivo. However, TEVV can play some physiological functions in 3 months in vivo, which can not suggest that TEVV can play long-term physical function in lower limb. In addition, the Institute of Professor.Zhang also conducted TEVV safety evaluation studies in vivo, the results showed that TEVV had no immune rejection response with the recipient animals, had no cellular toxic too, TEVV had good biocompatibility with the host animal. All these results suggested the TEVV constructed by implanted bone marrow-derived MAPC and the EPC into the allogenic acellular scaffolds were safe to recipient animal.Up to now, TEVV research has made great progress, but there are many problems need to be solved. Facing all above problems, this subject tried to use sheep allogeneic acellular vein graft as scaffold material, then combined multi-point injection and adding pressure perfusion technique, respectively implanted adult multipotent progenitor cells (MAPC) and endothelial progenitor cells (EPC) into scaffold material, at last construct Tissue Engineering venous valve in vitro, which we called "sheep MAPC / EPC TEVV "; Next, we anastomosed TEVV to the recipient sheep femoral vein using end to end method, and carried on long time function observation in vivo. We respectively applied color Doppler ultrasound, small animal ultrasound, digital subtraction imaging instrument to investigate the effectiveness of TEVV, at last, in different time point, all the transplanted TEVV and accelular scaffolds were harvested, we used HE staining, immunohistochemistry, scanning electron microscopy, transmission electron microscopy to detect the seeding cells distribution, growth, differentiation and evolution situation in the scaffold material. Our purpose was to research successfully a kind of TEVV which can be applied for long time in vivo so that TEVV can be used to treat chronic venous insufficiency in clinic. Part I. Tissue-Engineered Vein Valve Construction in VitroObjective: Implanted MAPC and EPC derived of sheep bone marrow into acellular venous scaffold containing venous valve and used multi-point injection and adding pressure perfusion method to construct tissue engineering venous valve. Our purpose is to provide a basic experimental evidence for tissue engineering venous valve's long-term research in vivo.Materials and Methods: MAPC and EPC primary culture was done, they were from bone marrow of sheep posterior superior iliac spine, MAPC and EPC passage cells were selected by CD45- and CD133+ using magnetic bead separation method respectively; After cell sorting, MAPC and EPC were detected SSEA-1, CD13 and CD34, CD133 by FLC respectively; Using GADPH as control, MAPC's markers OCT-4, SSEA-1 and EPC's markers KDR, VE-cadherin, eNOS were detected by RT-PCR respectively ; MAPC for CD13, SSEA-1, CD44, CD45, MHC-‖and EPC for CD133, CD34, VWF were detected respectively by using immune cell fluorescent chemical methods. Acellular venous scaffolds containing venous valve were gained by utilizing 0.5% Triton-100 +0.05% NH4OH, then digested with DNase + RNase enzymes; Cells, elastic fibers and collagen fibers inside acellular scaffolds were observed using HE staining and van Gieson staining; MAPC were marked with Hochest and EPC were marked with PKH26 after separation and amplification; Used multi-point injection and adding pressure perfusion method to implant MAPC and EPC into acellular scaffold to construct tissue engineering venous valve in vitro, then observed MAPC's and EPC's distribution and migration inside scaffold by fluorescence microscope after frozen section; The same method was used to implant not marked sheep bone marrow drived MAPC and EPC into scaffold to construct tissue engineering venous valve. The differences of the cells distribution in vascular wall were observed among constructed tissue engineering venous valve , acellular scaffold, natural veins by using scan electron microscopy; The differences of blood vessels elastic fracture intensity among constructed tissue engineering venous valve , acellular scaffold, natural veins were detected by elastic recovery test. Seeding cells distribution in constructed tissue engineering venous valve were detected by HE staining; Expression for Desmin,α-actin of SMC and VWF, VEGFR, CD133 of EPC were detected using immunohistochemical fluorescence method in constructed tissue engineering venous valve .Results: Sorted CD45- MAPC and CD133+ EPC morphology uniform, triangular or long spindle-shaped, two kinds of cells have strong proliferative ability, cells spread like cobblestone after confluence. After cells were sorted, through flow cytometry analysis showed: about positive rate, MAPC's SSEA-1was 31.47%, CD13 was 21.19%, EPC's CD34, CD133 was 57.69%, 53.63% respectively; immunofluorescence staining showed: MAPC express CD13, SSEA-1, but not express CD44, CD45, MHC-Ⅱ; EPC express CD133, CD34, VEGFR. RT-PCR results showed: MAPC express OCT-4, SSEA-1; EPC express KDR, VE-cadherin and eNOS. MAPC and EPC present blue and red through fluorescence microscope after being marked with Hochest and PKH26 respectively, after using marked MAPC and EPC to construct tissue engineering venous valve, through fluorescence microscopy after the frozen section showed the marked seed cells grew in acellular scaffolds and on both sides of valves leaflet and formatted cell monolayer. Compared with natural blood vessels, constructed tissue engineering venous valves have follow results: 1)Cells'distribution in blood vessel wall is similar through scanning electron microscope; 2)About elastic fracture strength, there was a little difference between constructed tissue engineering venous valve and natural blood vessels, but it was significant difference between constructed tissue engineering venous valve and acellular scaffold; 3)HE staining showed no cells remained in acellular scaffold, cells arranged regularly in natural blood vessel, there were cells monolayer on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; 4)Van Gieson staining showed the distribution of elastic fibers and collagen fibers was similar among acellular scaffold, constructed blood vessels containing tissue engineering venous valve and natural blood vessels; 5) immunohistochemical staining showed Desmin,α-actin, VWF, VEGF, CD133 were positive on tissue engineering venous valve.Conclusion: MAPC and EPC can be successfully cultured from sheep bone marrow; combination application of MAPC and EPC, using multi-point injection and adding pressure perfusion method to implant seeding cells into the acellular scaffold, then in vitro constructed tissue engineering venous valve which is similar to natural blood vessel wall structure; implanted seeding cells can spread successfully monolayer and endothelializate on both sides of constructed tissue engineering venous valve and inner surface of blood vessel wall; All these results provide experimental foundation for tissue engineering venous valve's long-term research in vivo the next. Part II. In vivo investigation of MAPCs/EPCs tissue engineered venous valvesObjective: To investigate physiological function and it's lasting time of TEVV by sheep MAPCs/EPCs TEVV long time (one year) effective observation in vivo and to provide a reliable experimental basis and theoretical basis for the application of TEVV on treating deep venous insufficiency in clinic in the future.Materials and Methods: 12 female sheep (age 1 y), divided into 3 groups, n = 4/group. The sheep TEVV were anastomosised to the right femoral vein of recipient sheep as the experimental group. The acellular scaffold were anastomosised to the left femoral vein of recipient sheep as the control group. In 3, 6,1 2 months after operation, respectively using color Doppler ultrasound, small animal ultrasound and digital subtraction angiography (DSA) to detect the function of TEVV; At last, the sheep were sacrificed and TEVV were taken out in 3,6,12, respectively, after general observation, the TEVV were fixed by 4% paraformaldehyde, and were embedded in paraffin, then paraffin sections were detected by using HE and immunohistochemical staining, scanning electron microscopy and transmission electron microscopy.Results: Efftective investigation results were followed: color Doppler ultrasound examination showed that postoperative patency rate of experimental group (control group) were 4/4 (4/4) in 3 months, 4 / 4 (1 / 4) in 6 months, 1 / 4 (0 / 4) in 12 months; In 3 months after operation, DSA detection showed there no reflux in the experimental group and significant reflux in control group; In 6 months after operation, small animal ultrasound showed there was valves resonance in experimental group, but valve's movement is limited, the control group vascular wall was thicker than experimental group and valve's shape was incomplete. In general, transmission electron microscopy, scanning electron microscopy, histological and immunohistochemical detection showed: the experimental group in 3 months after operation, no thrombosis, no obvious being thicken on vascular wall and surface of valves, were similar to the shape of normal valves, on the surface of valves and endomembrane, VWF and VEGFR were positive; VE-cadherin,α-actin and Desmin were positive on the tunica media. However, the control group had visible thrombus, HE staining showed that there were larger thrombus on the valves, the expression of VEGFR, VWF,α-actin, VE-cadherin and Desmin were evident; For the experimental group, in 6 months after operation, there was light thrombus near the valve, but no obvious being thicken in the vascular wall, valve morphology could be clearly recognized, VEGFR and VWF were positive expression in the endomembrane,α-actin and Desmin were positive too in the tunica media; For the experimental group, in 12 months after operation, venous valves and vascular wall were significantly thicker, there were visible thrombus, but valve morphology could still be identified. VEGFR, VWF of endomembrane andα-actin, VE-cadherin and Desmin of tunica media were expressed positively; For the control group, venous valve was thickened to be similar to the vascular wall and it's three-layer structure could not be distinguished, in 3 months after operation,α-actin, VE-cadherin and Desmin were positive in valves and endomembrane, and in 6,12 months after operation, valve's wall were extremely thickened that lumen were blocked by thrombosis, valve's structure was destroyed and valve's morphology could not be recognized clearly, thrombosis were obvious.α-actin, VE-cadherin, Desmin, VEGFR, VWF were not expressed.Conclusion: Sheep MAPCs/EPCs TEVV had valve function in six months after operation in vivo, but it's function was disappear in a year after operation. All above test results suggested that constructed TEVV in the current condition could not make up their function long time in vivo, the construction method needed to be further improved so that TEVV physiological function in vivo can be increased.