Construction of a vascularized bladder with autologous adipose-derived stromal vascular fraction cells combined with bladder acellular matrix via tissue engineering

Abstract

The formation of an effective vascular network can promote peripheral angiogenesis, ensuring an effective supply of blood, oxygen, and nutrients to an engineered bladder, which is important for bladder tissue engineering. Stromal vascular fraction cells (SVFs) promote vascularization and improve the function of injured tissues. In this study, adipose tissue-derived SVFs were introduced as an angiogenic cell source and seeded into the bladder acellular matrix (BAM) to generate a SVF-BAM complex for bladder reconstruction. The morphological regeneration and functional restoration of the engineered bladder were evaluated. In addition, we also explored the role of the Wnt5a/sFlt-1 noncanonical Wnt signaling pathway in regulating the angiogenesis of SVFs, and in maintaining the rational capability of SVFs to differentiate into vasculature in regenerated tissues. Histological assessment indicated that the SVF-BAM complex was more effective in promoting smooth muscle, vascular, and nerve regeneration than BAM alone and subsequently led to the restoration of bladder volume and bladder compliance. Moreover, exogenous Wnt5a was able to enhance angiogenesis by increasing the activity of MMP2, MMP9, and VEGFR2. Simultaneously, the expression of sFlt-1 was also increased, which enhanced the stability of the SVFs angiogenic capability. SVFs may be a potential cell source for tissue-engineered bladders. The Wnt5a/sFlt-1 pathway is involved in the regulation of autologous vascular formation by SVFs. The rational regulation of this pathway can promote neo-microvascularization in tissue-engineered bladders.

Keywords: Bladder augmentation; Wnt5a; bladder acellular matrix; stromal vascular fraction cells.

Figure 1.

SVFs extraction and identification: (a) Characterization of freshly isolated SVFs was performed by flow cytometry. The representative flow cytometry histogram of SVFs shows that freshly isolated SVFs could express hematopoietic, mesenchymal, and endothelial cell markers and (b) colony-forming unit (CFU) assay and staining.

Figure 2.

The rat subcutaneous transplantation model: (a) BAM seeded with SVFs was placed and fixed in the subcutaneous space in a rat, (b) BAM was placed and fixed in the subcutaneous space in a rat, and (c) photomicrographs of an H&E stained sample 14 days following transplantation. Representative microvessel structures were marked with arrows. Magnification ×200. *p < 0.05.

Figure 3.

CM-Dil-positive SVFs were clearly observed to be involved in bladder tissue reconstruction at 1 month after surgery, magnification ×100.

Figure 4.

Immunohistochemical assessments of regenerated bladders in all groups. Photomicrographs of urothelial markers (AE1/AE3) and a blood vessel endothelial marker (CD31) in all groups, at 1 and 3 months following transplantation. For all panels, magnification ×100. *p < 0.05; **p < 0.01. #No significant difference when compared with the BAM group.

Figure 5.

Immunohistochemical assessments of regenerated bladders in all groups: Photomicrographs of the smooth contractile muscle marker (α-SMA) post transplantation and Photomicrographs of the neuronal marker (S-100) post transplantation. Magnification ×100 in the α-SMA panels; magnification ×200 in the S-100 panels. *p < 0.05; **p < 0.01. #No significant difference when compared with the BAM group.

Figure 6.

Quantification of the urodynamic parameters at 3 months after bladder reconstruction. The bladder capacity (a) and bladder compliance (b) in the SVF group were significantly greater than those in the BAM group and were similar to those in the cystotomy groups. *p < 0.05; **p < 0.01. #No significant difference when compared with the cystotomy group.

Figure 7.

Exogenous recombinant Wnt5a mediates SVFs vascular self-assembly. SVFs were treated with increasing concentrations of recombinant Wnt5a: (a and b) The effects of various concentrations of recombinant Wnt5a on neovascularization in Matrigel plugs. After stimulation with different concentrations of recombinant Wnt5a, the plugs were removed on day 14 after Matrigel injection for the visualization and quantification of angiogenesis. Representative photographs of plugs from groups of five animals are shown. Quantification of angiogenesis within the Matrigel plugs is shown for all conditions. (c and d) During stimulation with different concentrations of Wnt5a, the expression of the vascularization-stimulating factors MMP2, MMP9, VEGFR2, and sFlt-1 in SVFs gradually increased. (e and f) At 4 weeks, the expression of Wnt5a and the vascular inhibitor sFlt-1 in the SVF-BAM group simultaneously increased. *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 8.

sFlt-1 suppresses the angiogenesis of SVFs: (a) Tube formation assays in SVFs treated with sFlt-1 neutralizing antibody, magnification ×40 and (b) Matrigel plug angiogenesis assay in SVFs treated with sFlt-1 neutralizing antibody. **p < 0.01.