A smart bilayered scaffold supporting keratinocytes and muscle cells in micro/nano-scale for urethral reconstruction

Abstract

Rationale: In urethral tissue engineering, the currently available reconstructive procedures are insufficient due to a lack of appropriate scaffolds that would support the needs of various cell types. To address this problem, we developed a bilayer scaffold comprising a microporous network of silk fibroin (SF) and a nanoporous bacterial cellulose (BC) scaffold and evaluated its feasibility and potential for long-segment urethral regeneration in a dog model. Methods: The freeze-drying and self-assembling method was used to fabricate the bilayer scaffold by stationary cultivation G. xylinus using SF scaffold as a template. The surface morphology, porosity and mechanical properties of all prepared SF-BC scaffolds were characterized using Scanning electron microscopy (SEM), microcomputed tomography and universal testing machine. To further investigate the suitability of the bilayer scaffolds for tissue engineering applications, biocompatibility was assessed using an MTT assay. The cell distribution, viability and morphology were evaluated by seeding epithelial cells and muscle cells on the scaffolds, using the 3D laser scanning confocal microscopy, and SEM. The effects of urethral reconstruction with SF-BC bilayer scaffold was evaluated in dog urethral defect models. Results: Scanning electron microscopy revealed that SF-BC scaffold had a clear bilayer structure. The SF-BC bilayer scaffold is highly porous with a porosity of 85%. The average pore diameter of the porous layer in the bilayer SF-BC composites was 210.2±117.8 μm. Cultures established with lingual keratinocytes and lingual muscle cells confirmed the suitability of the SF-BC structures to support cell adhesion and proliferation. In addition, SEM demonstrated the ability of cells to attach to scaffold surfaces and the biocompatibility of the matrices with cells. At 3 months after implantation, urethra reconstructed with the SF-BC scaffold seeded with keratinocytes and muscle cells displayed superior structure compared to those with only SF-BC scaffold. Principal Conclusion: These results demonstrate that the bilayer SF-BC scaffold may be a promising biomaterial with good biocompatibility for urethral regeneration and could be used for numerous other types of hollow-organ tissue engineering grafts, including vascular, bladder, ureteral, bowel, and intestinal.

Keywords: bacterial cellulose; bilayer scaffold; lingual keratinocytes; muscle cells; silk fibroin; urethral reconstruction.

Figure 1

Schematic illustration of the biosynthesis of bilayer SF-BC composites and the experimental design to fabricate TE urethra.

Figure 2

Structural and mechanical analyses of the SF-BC scaffold. (A) Digital photographs of the SF-BC scaffold. (B) Cross-sectional scanning electron microscopy (SEM) images of the SF-BC scaffold. (C-E) Representative SEM images of the architectures in the dense layer. (F-H) Representative SEM images of the architectures in the porous layer.

Figure 3

(A) MTT results from proliferation assays using lingual keratinocytes. (B) MTT results from proliferation assays using lingual muscle cells. The lingual keratinocytes and muscle cells grew well in the medium containing SF-BC, and the OD values in the scaffold groups were similar to those in the control group.

Figure 4

In vitro incubation of engineered urethral tissue. (A, C) Immunofluorescence staining of lingual keratinocytes and muscle cells. (B) Immunofluorescence staining of the SF-BC scaffold with DAPI. (D) Immunofluorescence staining of a cross-section of a cell-seeded SF-BC scaffold. (E) Immunofluorescence staining demonstrating that keratinocytes formed a compact confluent keratinocyte layer. (F) Immunofluorescence staining demonstrating that muscle cells invaded the SF-BC scaffold and dispersed throughout the porous polymer. (G-I) SEM analysis of keratinocytes on the compact surface of the SF-BC scaffold. (J-L) SEM analysis of muscle cells located inside the porous network that had grown along the SF wall. The white arrow indicates the cells, the yellow arrow indicates the nanofiber and the red arrow indicates the SF.

Figure 5

(A-F) During the surgical procedure in a dog model, the urethra between the bladder and the pubic symphysis was exposed, and a 5 cm long urethra section was transected and removed. Then, the scaffold was sutured onto the urethral defect. (G-L) Comparison of urethrography images in each group at 1 and 3 months after operation. The arrow indicates the urethroplasty site of the urethra.

Figure 6

Histologic analysis of reconstructed urethras at one and three months post-implantation. Evaluation of epithelium, smooth muscle and vessels with AE1/AE3, desmin and factor VIII immunohistochemical (IHC) staining in the retrieved urethra; H&E: hematoxylin and eosin.