J Korean Med Sci.  2020 Oct;35(41):e374. 10.3346/jkms.2020.35.e374.

Bladder Regeneration Using a Polycaprolactone Scaffold with a Gradient Structure and Growth Factors in a Partially Cystectomized Rat Model

• Kim HY
• Chun SY
• Lee EH
• Kim B
• Ha YS
• Chung JW
• Lee JN
• Kim BS
• Oh SH
• Kwon TG

• KMID: 2507829
• DOI: http://doi.org/10.3346/jkms.2020.35.e374

Abstract

Background
Tissue engineering can be used for bladder augmentation. However, conventional scaffolds result in fibrosis and graft shrinkage. This study applied an alternative polycaprolactone (PCL)-based scaffold (diameter = 5 mm) with a noble gradient structure and growth factors (GFs) (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) to enhance bladder tissue regeneration in a rat model.
Methods
Partially excised urinary bladders of 5-week-old male Slc:SD rats were reconstructed with the scaffold (scaffold group) or the scaffold combined with GFs (GF group) and compared with sham-operated (control group) and untreated rats (partial cystectomy group). Evaluations of bladder volume, histology, immunohistochemistry (IHC), and molecular markers were performed at 4, 8, and 12 weeks after operation.
Results
The bladder volumes of the scaffold and GF group recovered to the normal range, and those of the GF group showed more enhanced augmentation. Histological evaluations revealed that the GF group showed more organized urothelial lining, dense extracellular matrix, frequent angiogenesis, and enhanced smooth muscle bundle regeneration than the scaffold group. IHC for α-smooth muscle actin, pan-cytokeratin, α-bungarotoxin, and CD8 revealed that the GF group showed high formation of smooth muscle, blood vessel, urothelium, neuromuscular junction and low immunogenicity. Concordantly, real-time polymerase chain reaction experiments revealed that the GF group showed a higher expression of transcripts associated with smooth muscle and urothelial differentiation. In a 6-month in vivo safety analysis, the GF group showed normal histology.
Conclusion
This study showed that a PCL scaffold with a gradient structure incorporating GFs improved bladder regeneration functionally and histologically.

Keyword

Bladder Regeneration; Polycaprolactone Scaffold; Gradient Structure; Growth Factors

Figure

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  Fig. 1 Polycaprolactone-based scaffold with a gradient structure and incorporated growth factors. (A) Scanning electron microscopy photographs of the top/bottom surfaces and the cross-sectional morphology. (B) Loading amount of each growth factor (ng/sheet) and sustained release of the growth factors from the scaffold for 25 days.EGF = epidermal growth factor, VEGF = vascular endothelial growth factor, bFGF = basic fibroblast growth factor.

• 

  Fig. 2 In vivo analysis of morphological and histological aspects. (A) Gross images. The partial cystectomy (arrowhead), scaffold remnants (arrow), tightly sutured scaffold on the bladder at week 0, and restored bladder volume at week 12 are indicated. (B) Bladder volume measurements at weeks 4, 8, and 12 after the cystectomy operation. (C) Histological analysis with hematoxylin and eosin staining. Regenerated urothelial layers (U), connective tissues (C), blood vessels (V), smooth muscle bundles (M) and scaffold (S) were marked.(D-G) Immunohistochemistry with antibodies specific to α-smooth muscle actin to detect the smooth muscle bundle and blood vessels (D, arrows indicate newly-formed blood vessels); pan-cytokeratin to detect the urothelium (E, arrows indicate newly-formed urothelium at the border of the regenerated tissue); α-bungarotoxin to detect neuromuscular junctions (F, arrows indicate newly-formed neuromuscular junctions in the smooth muscle bundle); and CD8 to detect cytotoxic T cells (G, arrows indicate positively stained cells and the size of each arrow corresponds to the distribution of the cells).Control = sham-operated group, Partial cystectomy = the group with a 50% defect created by surgery, Scaffold = the group treated using the unmodified scaffold after partial cystectomy, GF = the group treated using the scaffold with incorporated growth factors (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) after partial cystectomy.*P < 0.05; **P < 0.01.

• 

  Fig. 3 In vivo analysis of gene expression. The smooth muscle (A) and urothelium (B) differentiation related gene expression.Control = sham-operated group, Partial cystectomy = the group with a 50% defect created by surgery, Scaffold = the group treated using the unmodified scaffold after partial cystectomy, GF = the group treated using the scaffold with incorporated growth factors (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) after partial cystectomy.MyoD = Myoblast determination protein, α-SMA = alpha smooth muscle actin, UP1a = uroplakin 1A, UP1b = uroplakin 1B, UP2 = uroplakin 2, CK7 = cytokeratin-7, CK13 = cytokeratin-13, CK18 = cytokeratin-18, CK19 = cytokeratin-19, PAN-CK = pan-cytokeratin, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.*P < 0.05; **P < 0.01.

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  Fig. 4 In vivo 6-month safety.GF = the group treated using the scaffold with incorporated GFs (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) after partial cystectomy.

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