| Prosthetic valve replacement was still the primary method to treat the heart valve diseases in the world now. It was consists of the mechanical and biological protheses valves. Its invention was a remedy for human beings. The survival quality of patients was improved enormously. But there were some insufficient accompaniments existed. Mechanical valves needed anticoagulation all rest of life and had associated with some risks of hemorrhage, thromboembolism and thrombogenesis; Biological valves had undurability and suffered from structural dysfunction because of calcification and progressive tissue deterioration. The point is valve prostheses basically represent nonviable structures and lack the potential to grow, to repair, or to remodel. Of particular importance to the pediatric population, homografts and all other currently available conduits and valves have no ability to grow. Only autografts (such as Ross valves transplanted from the pulmonary-to-aortic position in an individual) presently are viable, but the Ross procedure is technically difficult, risky, only serves a small patient subset, and has controversial results, including uncertainty over whether the grafts will grow commensurate with recipient growth. So, there is necessary to search a more consummate prostheses valve. The reseach of tissue engineering heart valve maybe resolve the problem and have make some advancement.Tissue engineering is an evolving science joining engineering and biology in an attempt to develop replacement tissue, and represents a novel scientific concept to overcome these limitations aiming at in vitro fabrication of living heart valves with a thromboresistant surface and a viable interstitium with repair and remodeling capabilities. As yet, there were some reports about successfully developing tissue engineered skin and tissue engineered bladder.At the present time, in the fields of studying tissue engineering heart valve, there are three key points existed including the best choice of the seeding cells and the sort of the scaffold and modified technology of recombination. Current options of seeding cells in tissue engineering heart valves are mostly coming from the vascular derived cells associated with certain shortcomings. Cell harvesting before seeding necessitated the sacrifice of intact vascular structures of the donor organism. Apart from that, vascular derived cells demonstrated different characteristics compared with natural valvular interstitial cells.The qualities might be vital to the development and long term function of TEHV. Bone marrow mesenchymal stem cells (BMSCs) can be obtained from isolating and expanding ex vivo, and differentiated to various cell phenotypes including osteocytes, chondrocytes, adipocytes, and myocytes etc. There are some research reports about endothelial cells (ECs) differentiation. Especially to take into account application in future routine clinical, we identified the goat BMSCs as the seeding cells. The first, they can be collected easily and consistent with mini-trauma concept avoiding adverse effect. Secondry, they have the potential to differentiate into multiple cell lineages and can growth and amplificate in the allogenic biomaterial scaffolds. In the micro- environment of the organism, it perhaps happened that the BMSCs could differentiate to the interstitial cells of vavle and ECs. We took the acellular porcine aortic valve as the scaffold of TEHV because it was the advantage of our research team previously. We especially introduce the polyethylene glycol (PEG) hydrogels technology to improve the recombination of the seeding cells with biomaterial scaffold to reconstruct TEHV ex vivo. The PEG-TEHVs had been implanted into the abdominal aorta of the cell donor goat to observe the results of the tissue formation and cell differentiation trendency in vivo environment. Further research and exploration had been practiced in the field of TEHV by our team.Part I: Isolation, purification, amplification and identification of the goat BMSCs: They were isolated and purified from the goat bone marrow by Percoll density gradient centrifugation and by adhering to the culture plastic. The morphology was observed under phase contrast microscope, and the some special antigens were examined by immunohistochemistry stain and identification was by the directional differentiation culture. Results: SH2 and Vimentin were positive expression in naturally differentiated BMSCs. CD34 and VIII related antigen were negative reaction.The results of be induced and differentiated to adipocyte cells showed positive staining of oil red. Intra-cell lipid droplets were seen obviously under the microscope. The resultes show that the cells obtained by our methods were identified to be BMSCs which had rich quantity and well activity. The 10～15 ml of marrow from a goat can provide a volume of (6.52±0.24)×107 cells after 4～6 passage in 20 days. It completely satisfied the seeding cells of the tissue engineering heart valve demands.Part II: Preparation of porcine acellular aortic valve and PEG-hydrogels: porcine aortic valves were decellularized with Triton X-100 and trypsin. We modified PEG-hydrogels with RGD, TGF-β1 and VEGF. BMSCs in modified PEG-hydrogels were seeded and grown in vitro in a static condition. Optimal precondition of the scaffolds was investigated in vitro. The activity effects of seeding cells were evaluated. Results: We found the BMSCs'biocompatibility of scaffolds were perfect by histology and scan electron microscopy (SEM). Preconditioning of scaffolds with modified PEG-hydrogels could make the BMSCs well-distributed and improve the efficacy of cell seeding[the percent of adhering of PEG-TEHV group vs Non-PEG group was (2.592±0.005 vs 1.885±0.004)]. We found an efficient approach of seeding cells to scaffolds and made a foundation for following study in vivo.Part III: Operation of transplantation into the abdominal aortas: Herein the goat's autologous BMSCs selected as the seeding-cells were encapsulated into the PEG-hydrogels to complete the process of the cells attaching to the acellular porcine aortic valves and the tendency of BMSCs'differentiation was observed when the mono semilunar TEHV had been implanted into their abdominal aortas. Gross and histology examination, light and electron microscopy observation were performed. PEG-TEHV was acted as group A; Non-PEG but substitute of media DMEM-LG and BMSCs were acted as group B; the goat's autologous aortic valve was selected as control group C. After 16 weeks, the samples were taken to exam for morphous, histology, SEM, transmission electron microscopy (TEM) and biomechanics test. We tried to make it clear that the direction of the BMSCs differentiation and the differences among the three groups. Results: Analysis of modified group A [PEG-hydrogels TEHV (PEG)] tensile strength, ratio of reendothelial (0.85±0.02:0.14±0.01) and mural thrombosis (0:100%) revealed much better improvement than the group B. The biomechanics of the group A got close to group C (goat native aortic heart valve). The data illustrated the critical importance of BMSCs's differentiation to endothelial and myofibroblast for remodeling into native tissue. Our results indicate that it is feasible to reconstruct TEHV efficiently by combining modified PEG-hydrogels with acelluar biomaterial scaffold and autologous BMSCs cells.
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