Bone marrow mesenchymal stem cells combined with acellular dermal matrix for repair of urethral injury

doi:10.3969/j.issn.2095-4344.0486

Abstract

Abstract:

BACKGROUND: In recent years, the development of tissue engineering provides more choices for the repair of urethral injury.
OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) combined with acellular dermal matrix in the repair of urethral injuries.
METHODS: Passage 3 BMSCs from New Zealand white rabbits were inoculated on the acellular dermal matrix to construct tissue-engineered urethra grafts. Thirty-six New Zealand rabbits were randomized into three groups (n=12 per group). Experimental group was implanted with BMSCs-acellular dermal matrix complex at urethral injury. Control group was implanted with acellular dermal matrix material at urethral injury. Normal group had neither injury nor treatment. At postoperative 4, 8 and 12 weeks, the repaired urethral tissue was subjected to hematoxylin-eosin staining. At postoperative 12 weeks, immunohistochemical staining and urodynamic study were performed.
RESULTS AND CONCLUSION: At postoperative 4 weeks, thin-layer epithelial regeneration was visible in the urethra defect area of the experimental group, and the continuity was better. The urethra mucosa of the control group was discontinuous. At postoperative 8-12 weeks, the urethral epithelial layer in the experimental group became thickened, exhibiting a good continuity with the normal urethral epithelium, thickened mucosa, and smooth and continuous urethral mucosa; the regenerated urethral mucosa of the control group exhibited good continuity, but less regenerated epithelial layers. At postoperative 12 weeks, immunohistochemical results showed the repaired urethra in the experimental group was positive for uroplakin IIIa, CK AE1/AE3, and α-smooth muscle actin. The maximum urethral pressure in the experimental group showed no significant difference before and after operation, while the postoperative pressure in the control group showed a significant increase (P < 0.05). Overall findings indicate that the combination of BMSCs and acellular dermal matrix has better efficacy than the acellular dermal matrix alone.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Urethral Diseases, Tissue Engineering

CLC Number:

R394.2

Fu Jin-shan, Zhou Zhi-yan, Xu Si-yi. Bone marrow mesenchymal stem cells combined with acellular dermal matrix for repair of urethral injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2075-2080.

Figures/Tables 2

2.1 骨髓间充质干细胞的形态骨髓间充质干细胞原代培养过程中，约24 h即可出现贴壁生长，细胞呈圆形、梭形、三角形，生长缓慢，换液后细胞增殖明显，出现以梭形为主的多种形态，10-14 d达70%-80%融合，可传代；传代细胞形态单一，呈梭形或扁平形，增殖至细胞融合时呈漩涡状和放射状排列(图1)。 2.2 骨髓间充质干细胞的鉴定 2.2.1 CD34、CD44表面标记物流式细胞仪检测结果显示，骨髓间充质干细胞表面标记物CD44阳性表达，CD34阴性表达(图2A)。 2.2.2 成骨诱导分化能力成骨诱导培养21 d，茜素红染色可见钙结节，呈阳性(图2B)。 2.2.3 成脂诱导分化能力成脂诱导培养21 d，油红O染色可见红色的脂滴颗粒，呈阳性(图2C)。 2.3 动物实验结果 2.3.1 大体观察结果术后4周，实验组脱细胞基质材料表面由黏膜覆盖，尿道表面光滑；对照组脱细胞基质表面无黏膜覆盖。随着时间的推移，术后12周时，实验组尿道黏膜表面无瘢痕形成，尿道及膀胱也未形成结晶；对照组尿道表面瘢痕明显，尿道及膀胱可见大量结晶。各时间点正常组尿道黏膜光滑平整。 2.3.2 苏木精-伊红染色观察结果见图3。术后4周，实验组尿道缺损区可见有少许薄层上皮再生，连续性较好；对照组修复段尿道新生黏膜尚不连续。术后8-12周，实验组修复段尿道上皮层逐渐变厚，与正常尿道上皮连续性良好，黏膜逐渐增厚，尿道黏膜光滑连续；对照组修复段尿道新生黏膜连续性较好，再生上皮层次较少。 2.3.3 免疫组织化学染色观察结果术后12周，免疫组化可见实验组修复尿道标本uroplakin Ⅲa阳性，CK AE1/AE3阳性。证实有成熟上皮细胞覆盖，且上皮细胞之间有连接功能。抗α-SMA免疫组化阳性证实新生尿道有排列规则的平滑肌束(图4)。 2.3.4 尿道造影检查结果实验组未发生尿道明显狭窄及尿瘘等并发症，对照组有实验动物出现尿道狭窄及尿瘘等并发症，但由于剩余实验动物数量较少，未进行统计学分析。 2.3.5 尿动力学检查结果实验组手术前后的最大尿道压比较差异无显著性意义；对照组手术前后的最大尿道压比较差异有显著性意义(表1)。"

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