Bladder Acellular Matrix Prepared by a Self-Designed Perfusion System and Adipose-Derived Stem Cells to Promote Bladder Tissue Regeneration

Abstract

The bladder patch constructed with the bladder acellular matrix (BAM) and adipose-derived stem cells (ASCs) was incubated with the omentum for bladder reconstruction in a rat model of bladder augmentation cystoplasty. A self-designed perfusion system and five different decellularization protocols were used to prepare the BAM. Finally, an optimal protocol (group C) was screened out by comparing the cell nucleus residue, collagen structure preservation and biologically active components retention of the prepared BAM. ASCs-seeded (BAM-ASCs group) and unseeded BAM (BAM group) were incubated with the omentum for 7 days to promote neovascularization and then perform bladder reconstruction. Hematoxylin and eosin and Masson's trichrome staining indicated that the bladder patches in the BAM-ASCs group could better regenerate the bladder wall structure compared to the BAM group. Moreover, immunofluorescence analyses demonstrated that the ASCs could promote the regeneration of smooth muscle, neurons and blood vessels, and the physiological function (maximal bladder capacity, max pressure prior to voiding and bladder compliance) restoration in the BAM-ASCs group. The results demonstrated that the self-designed perfusion system could quickly and efficiently prepare the whole bladder scaffold and confirmed that the prepared BAM could be used as the scaffold material for functional bladder tissue engineering applications.

Keywords: adipose-derived stem cells; bladder acellular matrix; perfusion system; tissue engineering; vascularization.

SCHEME 1

Schematic illustration on the preparation of the whole bladder scaffold by the self-designed perfusion system, and the tissue-engineered bladder patches incubated with the omentum for bladder augmentation.

FIGURE 1

Evaluation of decellularization efficiency of the different decellularization groups. (A) The composition and connection of the perfusion decellularization system. The arrow indicated the flow direction of the perfusion fluid. (B) H&E, DAPI, MTS and Alcian Blue staining of the native bladder tissue and the BAM prepared by different acellular groups. The DNA content (C), collagen content (D) and GAG content (E) of the native bladder tissue and the BAM prepared by different decellularization protocols. (F) Immunofluorescence staining of the BAM in group C with anti-collagen Ⅰ, anti-collage Ⅲ, anti-collagen Ⅳ, anti-fibronectin and anti-laminin. The SEM photomicrographs of the native bladder tissue (G) and the BAM (H) in group C. (B, F) Scale bar = 100 μM. (G, H) ×1000, scale bar = 10 μM. Significant difference is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

The growth and distribution of labeled ASCs in the BAM pieces and the vascularization of the constructed bladder patches incubated with omentum. (A) Live/dead detected the ASCs viability cultured in normal medium and the extract of BMA in group C. Green indicated the live cell. Red indicated the dead cell. (B) The cellular proliferative activity of the ASCs cultured in normal medium and the extract of BMA in group C. (C) The three-dimensional confocal photomicrographs of the labeled ASCs distributed on the BAM pieces. Red indicated the ASCs labeled with CM-DiI. (D) The SEM micrograph of the ASCs planted on the BAM pieces. (E) H&E and immunofluorescence staining of different bladder patches incubated with omentum for 7 days. The CD31-positive area was shown in green. White arrow: typical CM-DiI-labeled ASCs. The percentage of CD31-positive vessels area/total area (F) and the number of vessels (G) were compared among the different groups. (A, C) Scale bar = 200 μM. (D) ×1000, scale bar = 10 μM. (E) Scale bar = 100 μM. Significant difference is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Different bladder patches prompted bladder morphology restoration. Gross photographs of the bladder repair area in the BAM-ASCs group at 2 (A), 4 (B), 12 (C) weeks and in the BAM (D) and cystotomy (E) groups at 12 weeks. The retrograde cystography photographs of the bladder tissue in the BAM-ASCs group at 2 (F), 4 (G), 12 (H) weeks and in the BAM (I) and cystotomy (J) groups at 12 weeks. White arrows: labeled the stitch boundary of the bladder repair area. White triangles: marked the notch and diverticulum on the cystography images. Scale bar = 1 cm.

FIGURE 4

Histological evaluations of the regenerated bladder wall structure. Gross appearances of bladder tissue in the repair area and the regenerated areas of different groups (H&E). Histological staining of different structural layers of regenerated bladder wall in different groups, including urothelial layer (H&E), smooth muscle layer (H&E) and collagen tissue (blue in MTS). Dashed lines: margin of the repair area. UE: urothelial; LP: lamina propria; SM: smooth muscle. Gross photomicrographs: ×40, scale bar = 1 mm. Reg. area: ×100, scale bar = 200 μM. UE, SM and collagen: ×200, scale bar = 100 μM.

FIGURE 5

Immunofluorescence evaluations of the regenerated bladder tissue in the three groups. (A) Positive areas of AE1/AE3, α-SMA, NeuN and CD31 were showed in green (FITC), nuclei were indicated in blue (DAPI), and the ASCs were labeled in red (CM-DiI). Scale bar = 100 μM. The percentage per total field of AE1/AE3 (B), α-SMA (C), NeuN (D) and the vessel numbers (E) were quantitative analyses among the three groups. White arrows: CM-DiI-labeled ASCs. White triangles: showed the NeuN-positive neuron. Significant difference is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 compared with the cystotomy group.

FIGURE 6

Long-term functional evaluations of bladder tissue repaired with different bladder patches. Bladder capacity (A), peak pressure (B) and compliance (C) were tested at 12 weeks in the BAM-ASCs, BAM and cystotomy groups. Significant difference is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.