Construction of tissue engineered urethra by combining acellular matrix with exosomes in small intestinal submucosa
BACKGROUND:Small intestinal submucosal acellular matrix has been proven by clinical and basic studies to be useful for urethral repair and reconstruction.However,when applied alone,it has problems such as slow growth of host cells,difficulty in survival due to insufficient stent vascularization,and obstruction of reconstructed urethral stricture,and is only suitable for short urethral stricture.OBJECTIVE:To investigate the feasibility of constructing tissue engineered urethra with acellular matrix combined with exosomes in small intestinal submucosa.METHODS:Exosomes were isolated from rabbit bone marrow mesenchymal stem cells.The porcine small intestinal submucosal acellular matrix was prepared,and exosomes were loaded on the porcine small intestinal submucosal acellular matrix.The extracellular stroma-exosome(PKH26 dye labeled)complex of small intestinal submucosa was co-cultured with umbilical vein endothelial cells for 12 hours to observe the uptake of exosomes.The umbilical vein endothelial cells with good growth status were selected and cultured in three groups:the blank group was cultured routinely.The control group was added with small intestinal submucosal acellular matrix,and the experimental group was added with small intestinal submucosal acellular stromat-exosome complex.The angiogenesis was evaluated by scratch test,tube formation test,and angiogenic factor secretion test.Thirty New Zealand white rabbits were selected to establish a long(3 cm)urethral defect model,and the intervention was divided into three groups by random number table method(n=10).The single material group was implanted with small intestinal submucosal acellular matrix.The control group was implanted with small intestinal submucosal acellular matrix-bone marrow mesenchymal stem cell complex.The experimental group was implanted with small intestinal submucosal acellular matrix-exosome complex.Urethrography,urodynamic examination,and pathological observation of reconstructed urethral sections were performed 12 weeks after implantation.RESULTS AND CONCLUSION:(1)Exosomes in the acellular matrix of small intestinal submucosa could be taken up by umbilical vein endothelial cells under the fluorescence microscope.(2)Compared with the blank group and the control group,the experimental group could promote the migration of umbilical vein endothelial cells,angiogenesis ability,and the secretion of angiogenic factors vascular endothelial growth factor,hepatocyte growth factor,and interleukin 8(P<0.05).(3)Urethrography results showed that all 10 rabbits in the single material group had urethral stenosis;2 out of 10 rabbits in the control group had urethral stenosis,and none of the 10 rabbits in the experimental group had urethral stenosis.The results of urodynamic examination showed that the maximum urethral pressure at 12 weeks after implantation was higher in the single material group than before surgery(P<0.05),and the maximum urethral pressure at 12 weeks after implantation was lower in the control group and the experimental group than in the blank group(P<0.05).Hematoxylin-eosin,Masson and immunohistochemical staining showed that in the single material group,there were obvious regenerated epidermis,a small amount of subcutaneous smooth muscle and blood vessels,mainly fibrous tissue hyperplasia,accompanied by obvious inflammatory cell infiltration.In the control group,there were more complete regenerated epithelium and a small amount of collagen,a large number of subcutaneous blood vessels and smooth muscle,accompanied by inflammatory cell infiltration.The experimental group showed complete regenerated epidermis,a large number of subcutaneous blood vessels and smooth muscle,and no obvious inflammatory cell infiltration.The positive expressions of AE1/AE3,alpha smooth muscle actin,and CD31 in the experimental group were higher than those in the single material group and the control group(P<0.05).(4)The results show that the small intestinal submucosal acellular matrix-exosome tissue engineered urethra can repair the urethral defect by promoting angiogenesis.
万方数据
维普
