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Objective: To estimate the function of human bone marrow derived mesenchymal stem cells (hBMSCs) loaded on collagen scaffolds in the regeneration of canine tissue-engineered urethras. Methods:In this study, hBMSCs (1×10 6 hBMSCs per 1cm 2 collagen scaffold) were loaded on tubular collagen scaffolds (5cm in length). A 5cm long urethra segment near the penis bone was excised surgically. A collagen scaffold seeded with hBMSCs was sutured to two ends of the host urethra for the urethral repair in the experimental group, whereas the unseeded scaffold was used in the control group. Retrograde urethrography was performed before the operation and at 6 months after the surgery. At 6 months postsurgery, the animals were sacrificed, and the reconstructed urethras were collected for histological observation. An anti-human nuclei monoclonal antibody was used for immunofluorescent staining to detect the human cells in the reconstructed urethras. The growth of the urothelium, smooth muscles and blood vessels were evaluated by immunofluorescent staining. Results:The macroscopic observation and retrograde urethrography demonstrated that varying degree of urethra strictures were observed in all the animals of two groups. But the severer urethrostenosis usually appeared in the control group. Histologically, the scaffolds seeded with the hBMSCs formed a stratified urothelium layer that was similar to the appearance of the normal urothelium; however, in most areas, the unseeded scaffolds formed a discontinuous mucosa with a monolayer of urothelial cells. There were more smooth muscles and blood vessels growing than those in controls, although they were far fewer than normal. Positive cells were found in the experimental group with anti-human nuclei immunofluorescence staining. Conclusion:In this study, hBMSCs loaded on collagen scaffolds were able to promote the regeneration of canine tissue-engineered urethras, predominantly for the urothelium.