Studies On Fabricating Of Tissue Engineered Venous Grafts Based On Endothelial Progenitor Cells And Decellularized Aortic Matrices

Background: The concept of"Tissue Engineering", being introduced in the late of 1980s, anticipates tremendous clinical applicable potentials. To date, studies on tissue engineering of blood vessel (TEBV) are at the pre-clinical stage and have been focused mainly on arterial area. Few studies on tissue engineering of venous grafts (TEVG) had been reported.Objective: To explore the methods of fabricating TEVG in vitro based on canine autologous bone marrow-derived endothelial progenitor cells (EPCs) and xenologous (porcine) decellularized aortic matrices and to evaluate the feasibility and patency of the constructs after they are implanted into the same dog by replacing inferior vena cavas.Methods:(1) Canine bone marrow mononuclear cells (BMMNCs) were cultured using conditioned medium and coating with fibronectin (Fn). Morphology was observed using phase contrast microscope and the growth curve was constructed to evaluate the efficacy of proliferation. At different time points, positive staining of VEGFR-2, CD133 and factorⅧrelated antigen by immunocytochemistry methods and percentage of CD133,VEGFR-2 and the double-positive cells measured by flow cytometry were analyzed to identify the nature and the efficacy of expansion. The cellular ultrastructure was observed by transition electron microscope (TEM). Ingestion of 1,1'-dioctadecyl -3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetyl low density lipoprotein (Dil-ac-LDL) was tested to evaluate the cell function.(2) 0.1% trypsin and 0.01% EDTA were applied to extract cells from porcine aorta at 37℃and continuously vibrating state. The histology and ultrastructure of the decellularized aorta were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization. The xenologous decellularized patchs were implanted subcutaneously in canines and the explants were observed histologically to evaluate the immunogenicity at postoperative 1, 3 and 6 weeks respectively. Seeding endothelial cells (ECs) from canine external jungular vein was also explored.(3) Histology and scanning electronic microscope (SEM) were applied to compare: seeding results of non-precoating and precoated with gelatin and Fn on the inner surface of the matrices; results of different rotary speed of dynamic seeding; results of static seeding and rotative seeding and results of single and multiple seeding EPCs on the inner surface of the matrices. Incorporation Dil-ac-LDL and anti-platelet adherence by the seeded EPCs were tested to evaluate functions of the seeded cells.(4) Based on the results of above experiments, autologous EPCs labeled with PKH26-GL and decellularized xenologous (porcine) arterial tubular matrices were used to fabricate TEVGs (n=8), which were implanted to replace autologous canine inferior vena cavas about 4cm long. Meantime one femoral artery-venous shunt about 1cm long was performed. The non-seeded matrices (n=4) were performed the same as control. Angiographies were performed and the TEVGs were explanted for gross observation, TEM, SEM, immunohistochemistry examinations and immuno- fluorescence examinations at postoperative 10 days, 4 weeks and 12 weeks respectively.Results:(1) Cluster-like attached cells grew to confluence at an average time of 10 days. The cells presented cobble stone-like appearance and could incorporate Dil-ac-LDL. VEGFR-2, CD133 and factorⅧrelated antigens on the cultured cells were positive by immunocytochemistry. An average of (1.3±0.3)×106cells could been expanded from 1ml of bone marrow. VEGFR-2 and CD133 double positive cells expanded 242 folds at 10th day measured by flow cytometry.(2) The fresh porcine aorta was completely removed of the cell components and the matrices were remained intact after decellularizing process for 96 hours. The thickness, stress-strain curve, UTS and SOF were not different significantly before and after decellularization. After implanting the acellularized patchs subcutaneously in canine, moderately lymphocytes and neutrophils infiltration were seen at the 1st week and fibroblasts composed majority of the infiltrated cells while accompanied neocapillaries formation at the 6th week. A monolayer was obtained when the ECs were seeded onto the inner luminal surface of the matrices, aligned the same direction on the whole.(3) A cell coverage rate of 84.4%±6.2% (n=12)was achieved by precoating Fn and single dynamic seeding EPCs with a rotative speed of 2.5 revolution per hour (rph). The EPCs incorporated Dil-ac-LDL on the tubular matrices were detected and platelets adherent to the inner luminal surface of matrices in seeded group were much less than the control group.(4) A mean time of 12 days was expended when the TEVGs were fabricated. The patent number of experiment (control) group were 7/7(2/4), 6/6(2/2)and 4/4(1/2)at postoperative 10 days, 4 weeks and 12 weeks respectively. At 12 weeks, tightly confluenced ECs which covered the whole inner luminal surface of the explants were detected by immunohistochemistry of factorⅧand SEM, while fibrin-based pseudointima was detected on the inner luminal surface of matrix in one dog from the control group. Meanwhile, fibroblasts andα-actin positive cells were found by TEM and immunohistochemistry. PKH26-GL labeled EPCs sustained on the luminal surface at a rather proportion accompanied by newly formed ECs. However, the explants in test group showed partial stenosis.Conclusions:(1) Ex vivo expansion can effectively expand EPCs from bone marrow,and the cell quantity expanded for 10 days could meet the requirements of TEBVs.(2) The decellularized porcine aortic matrices, treated by 0.1% trypsin and 0.01% EDTA, meet the requirements of TEBVs on biomechanical properties, immunogenicity and cell compatibility.(3) Better results can be achieved when seeding EPCs on the luminal surface of decellulared aortic matrices by precoated with fibronectin at a single and dynamic 2.5rph speed.(4) Such fabricated TEVGs based on autologous bone marrow-derived EPCs and deccellularized xenologous aortic matrices are promising. However, they should be further evaluated by altering the matrix and augmenting the sample size in a longer period of time.