Key Technich For Construction Of Tissue Engineered Venous Valve

Primary valve dysfunction is commonly seen in clinical practice. Venous valve transplantation is often considered as the last choice. However, autologous venous valve transplantation is unsatisfactory due to limited source. To develop a graft bearing immunologically tolerated tissue-engineered venous valve that will be incorporated into a native vessel and restore normal valve function for the treatment of deep venous insufficiency, a manufactured, tissue-engineered, nonimunogenic venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of deep venous insufficiency.Currently, studies in tissue engineered venous valve are still at the primary stage. In2003, 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 could not grow into the walls of scaffolds. After being replanted, the grafts could not undertake the long term function. Teebken et al.(2009) took the great saphenous vein endothelial cells as seed cells, the decellularized great saphenous vein as scaffolds to construct tissue engineered venous valves. After three-dimensional culture for8days, they found that cells could grow on the valve and on both sides of the vessel wall. However, this study did not plant cells in the vessel wall in transplantation in vivo.With the support of national, army and Shanghai municipal government foundations, our laboratory has been focusing on the study of tissue-engineered venous valves in recent years. We have used canine bone marrow-derived multipotent adult progenitor cells (Multipotent Adult Progenitor Cells, MAPC)/endothelial progenitor cells (Endothelial Prigenitor cells, EPC) as seed cells, and sheep acellular venous valve stents as scaffolds to successfully construct the tissue engineered vein, and test its functions in dogs and sheep in vivo. However, the long term function of venous valve in vivo is poor. We believe that this may be related to the seed cells, scaffolds, implantation methods of seed cells in the scaffolds, etc. It needs further study on the technologies of tissue engineered venous valve construction to improve the quality of tissue engineered venous valve and shorten the gap with the physiologic vein valve. In seed cell research, Teebken et al. selected peripheral mature cells, which is not suitable due to the cell activity. We selected bone marrow-derived stem cells as seed cells with desirable cell activity. However, the seed cell isolation and culture are complicated and require bone marrow blood culture and immunomagnetic bead selection, which is time-consuming, expensive and easily contaminated during the cell separation.In scaffold material preparation, the previous studies applied two different methods of acellular scaffold preparation. Which method is better has not been studied. The recently reported freezing and thawing+enzyme method was found to have little damage on acellular scaffolds in other tissues decellularization. But it is rarely applied in valve decellularization.In seed cells implantation, the cell adhesion on blood vessels and valve surface is not enough, the seeded cells are easily to fall off, which remain the difficulty in the construction of tissue engineered vessels and valves and constrain the development of cardiovascular tissue engineering.In this study we aim to resolve the problem in tissue-engineered veinous valve, to simplify the seed cells induced cultivation method, to obtain good activity, high purity seed cells; to obtain a good performance cell scaffold materials that structure damage little and decellariat completely, And the development of smooth muscle cell planting implement, improving the smooth muscle cell adhesion rate in the three aspects of the research; In order to improve the construction of tissue-engineered venous valve, and to improve tissue engineering venous valve long-term performance.Part Ⅰ. Rabbit bone marrow-derived EPCs and SPCs cultureObjective:To isolate and culture rabbit bone marrow-derived EPCs and smooth muscle progenitor cells (SPCs) to study their biological properties and assess the possibility as the seed cells for tissue-engineered venous valves.Materials and Methods:Density gradient centrifugation was used to obtain bone marrow blood mononuclear cells, which were sedimentated and cultured with EGM-2complete medium containing5%FBS to be induced to EPC and were cultured with EBM-2medium without VEGF containing5%FBS,20ng/ml PDGF-BB for SPC induction. First medium fluid update was carried out48h later and cell of the3rd generation underwent cryopreservation and recovery and the cell activity was measured before and after the cryopreservation. The cell morphology was observed under phase contrast microscopy and ultrastructures were observed under transmission electron microscopy. EPCs/SPCs surface marker-positive rates were detected7days and14days after the induction with immunofluorescence and flow cytometry. The uptake of DiI-ac-LDL and binding to FITC-UEA-1as well as vessel genesis on MatriGel were measured.Results:Biological features of EPS:EPCs were cultured for10days and the cells fused as monolayer, showing a "stepping stone" appearance. EPC expressed CD34, VEGFR-2and weakly expressed CD133. Under the transmission electron microscope, WP bodies could be seen within the EPCs cytoplasm. Biological functions detect showed visible EPCs grew on the Matrigel in a blood vessel-like form and could uptake DiI-ac-LDL and bind to FITC-UEA-1. No significant changes in cell growth before and after recovery. Biological features of SPCs:SPCs was cultured for14days and showed specific features of the vascular smooth muscle growth, namely,"peak-valley" growth way. SPCs expressed CD34and SMA without Ⅷ and VEGF-2expression. Myofilaments, parallel with the cell longitudinal axis, could be seen under the transmission electron microscope. SPCs could not uptake DiI-ac-LDL and bind to FITC-UEA-1and did not form vessel-like structures on the Matrigel.Conclusion:Mononuclear cells could be obtained through density gradient centrifugation of the bone marrow blood, which could be cultured and induced to EPCs and SPCs of high purity. SPCs could naturally differentiate into smooth muscle-like cells, without the need to induce MAPCs to differentiate into smooth muscle cells. Compared to separate isolation, culture and differentiation induction of EPCs and MAPCs, this was time-saving, economic and not easily contaminated. Part Ⅱ. Preparation of tissue-engineered venous valve acellular scaffolds with different methodsObjective:To compare the biological features of tissue-engineered venous valve acellular scaffolds constructed with3methods Materials and Methods:To prepare tissue-engineered venous valve acellular scaffolds with3methods:Sodium deoxycholate group:The veins with valves from Beagle dogs were immersed in the4%sodium deoxycholate, oscillated at4℃for1h for decellularization and then repeatedly washed with37℃50mL normal saline. The acellular veins with valves were obtained and stored in4℃PBS.Triton group:The veins with valves from Beagle dogs were immersed in0.5%Triton-100+0.05%of NH40H solution and oscillated at4℃for3d, shaken in ultrapure water at4℃for3d, treated with DNase+RNase treatment (37℃) for12h, washed with ultrapure water and disinfected with60CO irradiation and preserved at-80℃.Freezing and thawing+enzyme group:Scaffold materials in each group were randomly selected. HE staining was performed to observe the microstructure, a scanning electron microscope was used to observe the surface and internal ultrastructure, TEM plus DAPI staining were used to detect DNA residues, in vitro EPCs cell cultivation was used to examine cell compatibility and acellular scaffolds were subcutaneously embedded to test the histocompatibility.Results:These three methods all could completely remove the cells. DAPI fluorescent examination showed no residual DNA components in the scaffolds. HE staining and scanning electron microscopy showed that collagen fibers were arranged orderly and there were no significant collagen fiber structure changes in the freezing and thawing+enzyme group. Collagen fiber breakage and structural disorder were detected in other two groups. Compared with other two groups, in the freezing and thawing plus enzyme group, subcutaneously embedded scaffolds showed less infiltration of inflammatory cells less and better EPCs adhesion to the scaffolds.Conclusion:Repeated freezing and thawing combined with osmotic pressure changes and low concentrations of trypsin, ribonuclease could thoroughly remove cells in the rabbit vein with valves, preserve relatively complete extracellular matrix and maintain good tissue and cell compatibility, indicating that is was an ideal method for decellularization. Part Ⅲ. Smooth muscle planting device for construction of tissue engineered venous valveObjective:To develop a smooth muscle planting device to seed SPCs and EPCs, elevate cell planting rate and establish tissue-engineered venous valves with good functions.Materials and Methods:To develop a smooth muscle planting device SPCs/EPCs of the3rd generation were selected as the seed cells. SPCs were seeded with the device in the experimental group and were implanted with the pressurized perfusion multi-point injection method in the control group. After3days, EPCs were planted with the pressurized perfusion multi-point injection method and were cultured for4days. After4hours, the SPCs were made into frozen sections, and DAPI was used to observe SPCs planting density.24h scanning electron microscopy and toluidine blue staining were used to monitor the implanting condition of SPCs on the scaffolds. MTT assay, cell adhesion assay were used to detect the cell proliferation performance. A week after HE staining, immunohistochemical staining was used to observe the histological structure of the constructed tissue-engineered venous valves.Results:Four hours after the implantation, DAPI staining showed uniform density of SPCs in the experimental group and uneven density in the control group with local cell aggregation. Scanning electron microscopy showed that more cells adhering to the extravascular surface in the experimental group with a small amount of extracellular matrix secretion and a smooth surface of the scaffolds. In the control group, a small number of cells adhered to the scaffolds, which was honeycombed. Toluidine blue staining showed that more SPCs adhered to the scaffolds in the experimental group with higher cell density in the experimental group. MTT and cell adhesion experiments showed that cells in the experimental group had higher proliferative activity and the difference was statistically significant compared with the control group.Conclusion:The smooth muscle planting device worked in effectively and evenly planting SPCs, which then secreted extracellular matrix and restore the surface structure of the scaffolds, thus promoting the adhesion and proliferation of EPCs...