Construction of tissue engineered urethra by combining acellular matrix with exosomes in small intestinal submucosa

doi:10.12307/2025.086

Abstract

Abstract: 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.

Key words: urethral injury, urethral stenosis, small intestinal submucosal acellular matrix, extracellular matrix, exosome, tissue engineered urethra

CLC Number:

Wang Dan, Zhu Xiaojun, Li Zhicheng, Li Na. Construction of tissue engineered urethra by combining acellular matrix with exosomes in small intestinal submucosa[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(23): 4907-4914.

Figures/Tables 5

2.1 外泌体的形态与特征蛋白表达透射电镜下可见提取的外泌体呈结构完整的圆形囊泡，符合外泌体的形态特征(图1A)，纳米粒子跟踪分析囊泡直径大小主要集中在30-150 nm范围内(图1B)，Western blotting检测显示提取的外泌体CD63、CD9蛋白呈阳性表达，PBS中CD63、CD9蛋白呈阴性表达(图1C)。 2.2 小肠黏膜下层脱细胞基质的形态扫描电镜下可见小肠黏膜下层脱细胞基质呈现三维网格状结构，富含排列整齐的胶原纤维(图2A)，苏木精-伊红染色显示相较于脱细胞处理前(图2B)，小肠黏膜下层脱细胞基质已经没有细胞与血管成分，仅含有胶原纤维成分(图2C)。 2.3 小肠黏膜下层脱细胞基质-外泌体复合物的体外促血管生成作用 2.3.1 外泌体示踪实验荧光显微镜下可见脐静脉内皮细胞胞质出现红色荧光(图3)，表明小肠黏膜下层脱细胞基质中的外泌体可被脐静脉内皮细胞摄取，进而发挥其生物学效应。 2.3.2 脐静脉内皮细胞迁移实验划痕实验显示，与空白组相比，小肠黏膜下层脱细胞基质可促进脐静脉内皮细胞的迁移；与小肠黏膜下层脱细胞基质相比，小肠黏膜下层脱细胞基质-外泌体复合物可进一步促进脐静脉内皮细胞的迁移(图4)。定量分析结果显示，空白组、对照组、实验组伤口闭合面积分别为(16.28±2.06)%，(25.78±6.52)%，(45.38±8.12)%，3组间比较差异有显著性意义(F=36.214，P=0.015)。 2.3.3 脐静脉内皮细胞成管实验 3组细胞的管状结构显微镜图片见图5，均可见管状结构形成，其中小肠黏膜下层脱细胞基质-外泌体复合物组管状结构最多。利用Image J软件测量管状结构中的网格数量、主干数量与节点数量，见表1，实验组网格数量、主干数量与节点数量均多于对照组、空白组(P < 0.05)。"

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