Cell-seeded tubularized scaffolds for reconstruction of long urethral defects: a preclinical study

Abstract

Background: The treatment options for patients requiring repair of a long segment of the urethra are limited by the availability of autologous tissues. We previously reported that acellular collagen-based tubularized constructs seeded with cells are able to repair small urethral defects in a rabbit model.

Objective: We explored the feasibility of engineering clinically relevant long urethras for surgical reconstruction in a canine preclinical model.

Design, setting, and participants: Autologous bladder epithelial and smooth muscle cells from 15 male dogs were grown and seeded onto preconfigured collagen-based tubular matrices (6 cm in length). The perineal urethral segment was removed in 21 male dogs. Urethroplasties were performed with tubularized collagen scaffolds seeded with cells in 15 animals. Tubularized constructs without cells were implanted in six animals. Serial urethrography and three-dimensional computed tomography (CT) scans were performed pre- and postoperatively at 1, 3, 6, and 12 mo. The animals were euthanized at their predetermined time points (three animals at 1 mo, and four at 3, 6, and 12 mo) for analyses.

Outcome measurements and statistical analysis: Statistical analysis of CT imaging and histology was not needed.

Results and limitations: CT urethrograms showed wide-caliber urethras without strictures in animals implanted with cell-seeded matrices. The urethral segments replaced with acellular scaffolds collapsed. Gross examination of the urethral implants seeded with cells showed normal-appearing tissue without evidence of fibrosis. Histologically, an epithelial cell layer surrounded by muscle fiber bundles was observed on the cell-seeded constructs, and cellular organization increased over time. The epithelial and smooth muscle phenotypes were confirmed using antibodies to pancytokeratins AE1/AE3 and smooth muscle-specific desmin. Formation of an epithelial cell layer occurred in the unseeded constructs, but few muscle fibers formed.

Conclusions: Cell-seeded tubularized collagen scaffolds can be used to repair long urethral defects, whereas scaffolds without cells lead to poor tissue development and strictures. This study demonstrates that long tissue-engineered tubularized urethral segments may be used for urethroplasty in patients.

Fig. 1

Study overview. Autologous urothelial and smooth muscle cells, isolated from the bladder, are seeded on tubularized acellular collagen scaffolds for urethral replacement. The animals were followed for up to 12 mo for analyses.

Fig. 2

(a) Collagen-based scaffolds derived from bladder were chemically treated to remove cells. (b) The processed collagen-based scaffolds show absence of cells. Hematoxylin and eosin (H&E) staining showed no presence of cells after the decellularization process. (c) Tubularized urethral graft seeded with cells prior to surgery. (d) Implanted tubularized urethral graft at the time of surgery.

Fig. 3

(a) Bladder epithelial and (b) smooth muscle cells are grown and expanded in culture. Both the urothelial and smooth muscle cells show expression of (c) AE1/AE3 and (D) desmin, respectively.

Fig. 4

All scaffolds seeded with cells showed attachment of multilayered urothelial cells in the inner layer and muscle cells on the outer surface of the construct. The labeled cells survived after implantation in vivo. H&E = hematoxylin and eosin.

Fig. 5

(Top panel) Two-dimensional (2D) computed tomography (CT) urethrocystography; (bottom panel) three-dimensional reconstructed images. Animals that were treated with tubularized matrix without cells showed strictured urethra with fistula. Animals treated with cell-seeded tubularized matrix showed wide patent urethras at 12 mo after surgery.

Fig. 6

Histologic analysis of reconstructed urethras. Extensive fibrosis and scarce smooth muscle were seen in animals treated with matrix without cells. Animals treated with cell-seeded matrix show formation of complete layers of transitional epithelium and well-developed smooth muscle. H&E = hematoxylin and eosin.