J Korean Med Sci.  2015 Dec;30(12):1754-1763. 10.3346/jkms.2015.30.12.1754.

Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model

• Lee JN
• Chun SY
• Lee HJ
• Jang YJ
• Choi SH
• Kim DH
• Oh SH
• Song PH
• Lee JH
• Kim JK
• Kwon TG

• KMID: 2359959
• DOI: http://doi.org/10.3346/jkms.2015.30.12.1754

Abstract

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 microL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 microL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 microL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 microL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 microL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.

Keyword

Bladder Regeneration; Surface Modified Scaffold; Urine-derived Stem Cells; Basic Fibroblast Growth Factor 2

MeSH Terms

Adult Stem Cells/cytology/metabolism/*transplantation
Animals
Biocompatible Materials/chemistry
Cell Differentiation
Fibroblast Growth Factor 2/administration & dosage
Heparin/administration & dosage
Humans
Materials Testing
Models, Animal
Poloxamer
Polyesters
Rats
Reconstructive Surgical Procedures
Regeneration
Tissue Engineering/*methods
Tissue Scaffolds/chemistry
Urinary Bladder/anatomy & histology/physiology/*surgery
Urine/*cytology
Biocompatible Materials
Fibroblast Growth Factor 2
Heparin
Poloxamer
Polyesters

Figure

• 

  Fig. 1 Schematic diagram of the scaffold fabrication and operation procedures. (A) Procedures for fabrication of the heparin-immobilized bFGF-loaded scaffolds (Scaffoldheparin-bFGF) consisting of Polycaprolactone/Pluronic F127/bladder submucosa matrix (PCL/F127/BSM). (B) Bladder reconstruction operation procedure using the various scaffolds.

• 

  Fig. 2 Measurement of heparin, bFGF, and scaffoldheparin-bFGF biocompatibility. (A) Amount of immobilized heparin on the scaffold (n=3). (B) Visualization of immobilized heparin on the scaffold. (C) Affinity and amount of bFGF released from the heparin-immobilized scaffold for 28 days. An unmodified heparin scaffold was used as a control. (D) Field emission scanning electron microscope images of scaffold morphology and cells attached to the scaffold. (E) Cell adhesion to the scaffolds was determined by measuring the DNA concentration with Hoechst 33258 staining in confocal images. (F) Biocompatibility analysis of the scaffolds. Ctrl, culture plate dish; scaffold, unmodified scaffold; scaffoldheparin-bFGF, heparin-immobilized bFGF-loaded scaffold; USCs, urine derived stem cells; O.D., optical density. All data are presented as mean±SD (*P<0.01; †P<0.05).

• 

  Fig. 3 Cell differentiation supported by the scaffoldheparin-bFGF. (A) USC differentiation (%) into smooth muscle and urothelial cells on the scaffoldheparin-bFGF and representative FACS images. (B) Real-time PCR analysis of stem cell, myogenic, and urothelial lineage markers at days 0 and 14. *P<0.01.

• 

  Fig. 4 Morphological and immunohistochemical (IHC) analysis. (A) Morphological analysis of retrieved bladder. (B) Analysis of α-SM actin, pan-CK, CD8, and HuNu expression with IHC. α-SM actin, α-smooth muscle actin; pan-CK, pan-cytokeratin; CD8, cluster of differentiation 8; HuNu, Human nuclei-specific antibody. Control, sham operated; partial cystectomy, approximately 40% defect was created in the dome of the bladder wall; scaffold, unmodified scaffold was attached after partial cystectomy; scaffoldheparin-bFGF, the heparin-immobilized bFGF-loaded scaffold was attached after partial cystectomy; USC-scaffoldheparin-bFGF, scaffoldheparin-bFGF combined with USCs was attached after partial cystectomy. Scale bar=50 µm. Magnification, 400 ×.

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