Impact of fibrin glue and urinary bladder cell spraying on the in-vivo acellular matrix cellularization: a porcine pilot study.

PURPOSE: Urinary bladder tissue engineering utilizing autologous cell-seeded scaffolds requires enough bladder cells to populate a large surface area which may be difficult to obtain from abnormal bladders. We evaluated whether a fibrin glue spray technique enhances cell seeded acellular matrix (ACM) repopulation in a porcine bladder model. MATERIALS AND METHODS: Porcine urothelial and smooth muscle cells cultured from open bladder biopsy were sprayed with or without fibrin glue onto porcine bladder ACM. After 10 days in vitro, constructs were implanted onto porcine bladders (4/group) and harvested after 1 or 6 weeks for H&E and immunohistochemical staining. RESULTS: In vitro, fibrin glue was associated with more continuous cell growth and enhanced cellular organization, maintained particularly in the periphery in vivo, where both groups demonstrated central fibrosis. CONCLUSIONS: While fibrin glue enhanced cellular organization on ACM in vitro, central fibrosis in vivo suggests that factors supporting seeded cell survival are lacking.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for porcine bladder augmentation in vivo: a 6-week experimental study.

PURPOSE: In a pilot study we developed a cell transfer technology for populating demucosalized colonic segments with bladder urothelium. This process was achieved through aerosol transfer of a single cell suspension consisting of bladder urothelial cells, smooth muscle cells and fibrin glue onto demucosalized colonic segments. We further evaluate this new concept in a controlled study. MATERIALS AND METHODS: The study was performed on 20 piglets (20 kg). In all animals 50% of the bladder with excised, and a 10 cm segment of the sigmoid was isolated. Animals were then equally divided into 5 groups of 1) colocystoplasty only, 2) demucosalized colocystoplasty, 3) demucosalized colocystoplasty plus covering of the demucosalized sigmoid with fibrin glue only, 4) aerosol application of fibrin glue with single cell suspension of urothelial cells only to the demucosalized colon, and 5) aerosol application of fibrin glue with urothelial and smooth muscle cells to the demucosalized colon. The 4 corners of the augmented segments were marked with 5-zero polypropylene sutures. Animals were sacrificed 6 weeks later and the surface area of the augmented segment was measured. Segments were submitted to histological and immunohistochemical analysis. RESULTS: The surface area of the augmented segments showed an increase in group 1 animals, stabilization in groups 4 and 5, and marked reduction in groups 2 and 3. On hematoxylin and eosin, and Masson trichrome staining all group 1 animals showed normal colonic epithelium of the augment. All animals in groups 2 and 3 showed excessive scarring with urothelial coverage only at the augment periphery, while the central augment area showed no epithelium. Segments from groups 4 and 5 showed confluent epithelial covering with no fibrosis. There was no evidence of colonic epithelial re-growth in any animal in groups 2 to 5. Cytokeratin 7 and uroplakin III staining demonstrated complete coverage of the augmented segment with urothelium only in groups 4 and 5. CONCLUSIONS: The addition of aerosolized cells of urological origin is a viable augmentation approach that appears to achieve the much sought after inhibition of intrinsic fibrosis and contraction of colonic segments when incorporated into the urinary tract without this cellular component. Moreover, this technique appears to provide a histologically normal, confluent urothelium, which sets the stage for prevention of the well-documented biochemical aberrations inherent in augments containing gastrointestinal epithelium. While successful in this model regardless of the incorporation of urological smooth muscle cells, chronic studies are now warranted to validate the short-term results as well as determine whether the urological mesenchymal population (smooth muscle) will be required to sustain the uroepithelial phenotype in the long term.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments: a pilot study.

PURPOSE: We developed a cell transfer technology for covering demucosalized colonic segments with bladder urothelium. This covering would be achieved through aerosol spraying of single cell suspension of bladder urothelial and smooth muscle cells with fibrin glue onto the demucosalized colonic segments. MATERIALS AND METHODS: In 6 piglets (20 kg.) a 4 cm.2 area of bladder was excised. Single cell suspension of bladder urothelial and smooth muscle cells was prepared. A segment of detubularized sigmoid colon was isolated on its vascular pedicle and demucosalized. The single cell suspensions were combined with an equal volume of fibrin glue and sprayed over the raw submucosal surface of the sigmoid segment. The sigmoid segment was retubularized and sutured to the posterior peritoneum. Animals were sacrificed 4 weeks later, and the segment was submitted to histological and immunohistochemical analysis. RESULTS: Sigmoid segments appeared grossly intact with no reduction in surface area. Hematoxylin and eosin architecture revealed an intact urothelial layer. Deep to this layer was a randomly aligned but distinctly segregated layer of smooth muscle cells. The urological new smooth muscle layer stained positive for calponin and the urothelial layer was cytokeratin-7 and uroplakin III positive. CONCLUSIONS: Separation, cell suspension and aerosol delivery of bladder urothelial and smooth muscle cells in fibrin glue can successfully transfer these urological cell populations to a new host tissue commonly used in urological reconstruction. In vivo co-culture of bladder smooth muscle and urothelial cells results in coverage of a large area of demucosalized gut providing new potential for transfer and reconstitution of urologically functionally appropriate tissue to the bladder itself.