Adipose-derived stem cells combined with bladder acellular matrix for long-segment urethral defect repair: A preclinical study

Objective This study evaluated the feasibility of constructing tubular grafts using adipose-derived stem cells and smooth muscle cells seeded onto bladder acellular matrix scaffolds for repairing long-segment urethral defects. Methods A tissue-engineered urethra was constructed in dogs using autologous adipose-derived stem cells (ADSCs) and smooth muscle cells (SMCs) seeded onto bladder acellular matrix grafts (BAMGs). First, autologous ADSCs and SMCs were isolated and cultured separately from canine tissues via enzymatic digestion. ADSCs were characterized by flow cytometry and assessed for multi-lineage differentiation potential. Fresh canine bladder tissues were decellularized to prepare BAMGs. Expanded ADSCs were seeded onto one side of the BAMGs, and SMCs onto the other side. After one week of in vitro co-culture, the cell-scaffold constructs were rolled into tubular tissue-engineered urethras. Twenty-four male Beagle dogs were subjected to creation of a 4-cm anterior urethral defect and were randomly divided into experimental and control groups, with 12 dogs in each group. In the experimental group, the defects were repaired with the tissue-engineered urethra constructed from autologous ADSCs/SMCs and BAMGs, whereas the control group received BAMG alone. At 1, 2, 4, and 8 weeks postoperatively, tissue samples were harvested for evaluation of regenerative outcomes; at 8 weeks postoperatively, retrograde urethrography and cystourethroscopy were performed at the terminal assessment of each animal to assess the repair efficacy. Results ADSCs cultured in vitro were predominantly spindle-shaped and immunophenotypically characterized as CD90 + CD29 + CD34 − CD45 − , with confirmed adipogenic and osteogenic differentiation by Oil Red O and Alizarin Red S staining, respectively. ADSCs and SMCs seeded onto BAMGs showed favorable biocompatibility and growth.All experimental animals (n = 12) maintained patent urethras without stricture. In the control group, 2 animals died within the first 2 weeks, and of the 10 animals available for 8-week assessment, 6 developed overt urethral strictures. Experimental grafts displayed satisfactory epithelialization and smooth surfaces, while controls showed contracture, scarring, and calculi. Histologically, experimental animals exhibited well-organized multilayered urothelium and muscle with progressive regeneration over time, in contrast to the extensive fibrosis and scant smooth muscle seen in controls. At 8 weeks, cystoscopy and urethrography confirmed smooth mucosa and unobstructed lumens without extravasation in the experimental group, versus overt urethral collapse and obstruction in the control group. Conclusions These findings provide preclinical evidence that a tubularized scaffold based on bladder acellular matrix and co-seeded with ADSCs and SMCs is a promising strategy for repairing 4-cm circumferential urethral defects in a healthy canine model. Because this was an acute-defect study in an otherwise healthy urethra with follow-up limited to 8 weeks, the findings should be interpreted as a promising preclinical strategy rather than an established alternative to current autologous tissue-based procedures.