Urinary bladder smooth muscle engineered from adipose stem cells and a three dimensional synthetic composite

Abstract

Human adipose stem cells were cultured in smooth muscle inductive media and seeded into synthetic bladder composites to tissue engineer bladder smooth muscle. 85:15 Poly-lactic-glycolic acid bladder dome composites were cast using an electropulled microfiber luminal surface combined with an outer porous sponge. Cell-seeded bladders expressed smooth muscle actin, myosin heavy chain, calponinin, and caldesmon via RT-PCR and immunoflourescence. Nude rats (n=45) underwent removal of half their bladder and repair using: (i) augmentation with the adipose stem cell-seeded composites, (ii) augmentation with a matched acellular composite, or (iii) suture closure. Animals were followed for 12 weeks post-implantation and bladders were explanted serially. Results showed that bladder capacity and compliance were maintained in the cell-seeded group throughout the 12 weeks, but deteriorated in the acellular scaffold group sequentially with time. Control animals repaired with sutures regained their baseline bladder capacities by week 12, demonstrating a long-term limitation of this model. Histological analysis of explanted materials demonstrated viable adipose stem cells and increasing smooth muscle mass in the cell-seeded scaffolds with time. Tissue bath stimulation demonstrated smooth muscle contraction of the seeded implants but not the acellular implants after 12 weeks in vivo. Our study demonstrates the feasibility and short term physical properties of bladder tissue engineered from adipose stem cells.

Figure 1. Construction of the three dimensional synthetic bladder composite

A) Schematic and B) gross micrograph of the 3-dimensional bladder composite. C) PLGA electropulled microfibers comprising the luminal layer D) PLGA porous sponge was used as the outer layer.

Figure 2. Differentiation of ASCs into SM-ASCs

A) Myosin heavy chain (MHC) expression in SM-ASCs after 6 weeks incubation in SMIM (FITC conjugated; nucleus DAPI, 400x). B) Caldesmon expression (FITC, 400x)

Figure 3. Cell seeding of in vitro constructs

A) PLGA cell seeded bladder composite 14 days in vitro seeded with SM-ASCs labeled with DiI (red, 40x). B) Luminal edge of SM-ASC seeded scaffold 14 days in vitro stained with calcein AM (green) to demonstrate viable cells (100x).

Figure 4. Smooth muscle expression from SM-ASC seeded bladder constructs

A) RT-PCR and B) real-time RT-PCR of smooth muscle markers extracted from an SM-ASC seeded construct compared to undifferentiated ASCs on a scaffold. C. Caldesmon and D) MHC expression in an SM-ASC seeded scaffold (FITC conjugated, nucleus counterstained DAPI, 200x and 400x respectively).

Figure 5. Histology of cell seeded constructs after bladder augment

A — C) Gross appearance of tissue engineered implant after 3 days, 4 weeks, and 12 weeks respectively. D) Cross section of cell seeded augment at 8 weeks, anastamosis marked with an asterix, H & E, 10x. E) Same specimen under fluorescent microscope with SM-ASCs red (DiI), nuclei blue (Dapi), and urothelium green (FITC), reconstructed from 100x. F) Higher power demonstrating the anastamosis at 4 weeks (Mason’s Trichrome, 50x) and G) early formation of urothelium by 2 weeks, 200x.

Figure 6. Urothelium and smooth muscle in cell-seeded graft in vivo

A) Graft urothelium showing absence of DiI after 4 weeks in vivo (FITC; 200x) B) Early growth of smooth muscle bundles expressing MHC in an SM-ASC seeded (red) composite at 4 100x), C) Seeded SM-ASCs (red) after 12 weeks in vivo counterstained for smooth muscle myosin heavy chain (green). Areas with both marker appear yellow on image overlay (200x).

Figure 7. Comparison of SM-ASC and acellular grafts

A, B) DiI emitting SM-ASCs (red) are present only in the cell seeded scaffolds, nuclei counterstained with DAPI, 50x. C, D) Graft cores demonstrating cellular and collagen deposition in the micropores of the sponge, Mason’s Trichrome, 100x. E, F) Luminal electropulled surface showing smooth muscle and collagen deposition above a urothelial lining, 200x.

Figure 8. Physiologic bladder testing

A) Pressure/ volume curves of pre-operative and 2 week post-operative rat bladders. B) Animals with tissue engineered bladder augments maintained superior bladder volume and C) compliance over 12 weeks.

Figure 9. Pharmacologic smooth muscle contraction

A) Carbachol (1 × 10^-6 M to 1 × 10^-4 M) and KCl (80mM) induced contractions in native rat bladder (partial cystectomy group) and in augmented bladder explants from acellular and cell-seeded bladder constructs. B) Mean tissue contraction (± SEM) of the experimental and control bladder grafts after 12 weeks in vivo.