Effects Of Prevascularized Cell Sheets In The Vascularization Of The Decellularized Tracheal Scaffold

BackgroundCongenital tracheal stenosis is a rare and life-threatening disease.Tissue-engineered trachea is a new method for the treatment of severe tracheal stenosis,but the vascularization of tissue engineering trachea is difficult to solve,the transplantation fails due to insufficient blood supply.Prevascularization provides a new method to solve this problem.Umbilical vein endothelial cells co-cultured with cell sheets to form prevascularized cell sheets has been widely used in a variety of tissue engineering,and it has been proved to promote vascularization,but it has not been reported in the field of tissue engineering trachea.Objectives1.To construct the prevascularized cell sheets,and investigate its characteristics.2.To investigate the effects of prevascularized cell sheets in the vascularization of the decellularized tracheal scaffold.MethodsBMSCs were isolated and cultured by the method of the whole bone marrow adherent,and prepared the stem cell sheets by adding ascorbic acid.RUVECs and stem cell sheets were co-cultured to construct the prevascularized cell sheets.Decellularized trachea was prepared by an improved detergent-enzymatic method,and then combined with prevascularized cell sheets implanted subcutaneous to investigate its effects of vascularization.Results1.BMSCs were obtained from bone marrow of rabbit,growing adhering to the wall,showed spindle-shape.Flow cytometry showed that CD90 and CD44 were positive,but CD45 and CD34 were negative.Cells had the ability of multidirectional differentiation with osteogenesis and Adipogenesis,which showed that the cells had the ability of multidirectional differentiation.2.The cell sheets of BMSCs induced by ascorbic acid is mechanical and operable.Histological examination revealed that cell sheets consisted of multi-layered of cells and secreted ECM.RT-PCR results indicated that Col-1 and Fn were highly expressed in the cell sheets and were statistically significant(p<0.05).3.RUVECs were able to grow adhering to the wall.They are short fusiform,square or polygonal cells.When the cell density reaches more than 80%,the cell arrangement like a "paving stone".Immunological examination revealed that it has the characteristics of endothelial cells.4.RUVECs were co-cultured with stem cell sheets.Microscopic and histological observation showed that RUVECs were rearranged and migrated within 3 days.Immunohistochemical examination showed that prevascularized cell sheets showed vascularization.RT-PCR detected the mRNA expressions of vascularization related factors,VEGF,bFGF,Angl,and Ang2.The experimental group was higher than that in the control group and were statistically significant(p<0.05).5.HE and Masson staining showed that this trachea remove most of the cell components and nuclear substances and retain the original trachea structure.6.Subcutaneous transplantation of prevascularized cell sheet/decellularized tracheal scaffold.HE staining results subcutaneous transplantation can promote vascularization.The neovascularization area percentage of prevascularized cell sheet/decellularized tracheal scaffold group was higher than that of decellularized tracheal scaffold group and was statistically significant(p<0.05).Conclusions1.The prevascularized cell sheets with vascular network can be constructed in combination with RUVECs and stem cell sheets in vitro.2.The prevascularized cell sheets can promote the vascularization of decellularized tracheal scaffolds and provide a new method for promoting the vascularization of tissue-engineered trachea.