Autologous and heterotopic transplantation of adipose stromal vascular fraction ameliorates stress urinary incontinence in rats with simulated childbirth trauma

Abstract

Introduction: Autologous transplantation of adipose stromal vascular fraction (SVF) is a cost-effective and technically accessible option for cell therapy. Clinical study of SVF transplantation for male stress urinary incontinence (SUI) is underway, but the effectiveness remains unknown for female SUI, majority of which is caused by childbirth trauma.

Methods: Vaginal Distension (VD) rats were generated as in vivo model for female SUI. To quantitate the severity of SUI, leak point pressure (LPP) was measured by placing a bladder catheter. There was a characteristic waveform of LPP with two-peaks, and we counted the second peak as an LPP value. Adipose SVF was separated from inguinal fat and delivered into external urethral sphincter (EUS) through transperineal injection. LPP was measured 7 or 14 days after SVF transplantation. Tissue damage and collagen synthesis around the EUS were visualized by Masson's trichrome and eosin staining. Antibody against α-smooth muscle actin (α-SMA) was used to stain smooth muscle or activated stromal cells. Donor SVF cells were distinguished from recipient EUS tissue by tracking with GFP transgene.

Results: VD procedure decreased the frequency at which the normal LPP waveform appeared and lowered the LPP value. SVF injection normalized the waveform as well as the level of LPP. VD disrupted histological structure of EUS and SVF failed to differentiate into striatal muscles. Instead, SVF increased α-SMA positive cells and collagen synthesis but the phenomena depended on VD stimulus. GFP tracking indicated that the transplanted SVF cells persisted for four weeks and synthesized α-SMA protein simultaneously.

Conclusions: Autologous transplantation of adipose SVF displayed bulking effects through collagen synthesis. However, such heterotopic activation was dependent on tissue damage.

Keywords: Adipose stromal vascular fraction; Collagen synthesis; EUS, external urethral sphincter; LPP, leak point pressure; Leak point pressure; NPs, low-molecular-weight heparin/protamine micro/nanoparticles; PNT, pudendal nerve transection; SUI, stress urinary incontinence:; SVF, stromal vascular fraction; Stress urinary incontinence; VD, vaginal distension; Vaginal distension model rat.

Fig. 1

Experimental methodology used in this study. (a) Schematic diagram for the cell transplantation. Transperineal injection was performed around the urethral meatus and the depth was precisely adjusted, so that the SVF cells reach around the EUS. (b) Schematic diagram of the equipment for LPP measurement. (c) Representative waveforms of the LPP. Vertical scalebar: 100 mmHg. Horizontal scalebar: 30 s. U: urethra; Va: vagina; EUS: external urethral sphincter; LPP: leak point pressure; SVF: stromal vascular fraction; VD: vaginal distension.

Fig. 2

VD decreased LPP values, which heterotopic SVF cells normalized. (a) The scatter-plot for LPP values of 7 days after VD treatment (n = 6). (b) The scatter-plot for LPP values of 14 days after VD treatment (n = 10). *p < 0.05, **p < 0.01: significantly different between the test group (No Inj.) vs. control group (SVF or SVF + NPs). LPP: leak point pressure; VD: vaginal distension; SVF: stromal vascular fraction; NPs: low-molecular-weight heparin/protamine nanoparticle.

Fig. 3

VD disrupted external urethral sphincter and heterotopic SVF cells failed to restore the muscle fibers. Representative light micrographs of transverse sections of the urethras with immunohistochemistry against α-SMA and M-T plus eosin staining after VD treatment. Arrowheads indicate the approximate region where the disrupted sphincter could have existed. The images in the middle row are modified/edited by Adobe Photoshop CS6 for quantitative analysis (Green color: α-SMA immunoreactivity). Scale bar: 100 μm. α-SMA: alpha-smooth muscle actin; M-T: Masson's trichrome; VD: vaginal distension; SVF: stromal vascular fraction; NPs: low-molecular-weight heparin/protamine nanoparticle.

Fig. 4

Heterotopic SVF cells increased α-SMA positive cells and collagen synthesis around the damaged sphincter. (a) The percent of areas for α-SMA positive. (b) The percent of areas for collagen fibers (blue staining by M-T). The scatter plot of each percent pixels (n = 5). *p < 0.05, **p < 0.01: significantly different between the test group (No Inj.) vs. control group (SVF or SVF + NPs). SMA: smooth muscle actin; M-T: Masson's trichrome; VD: vaginal distension; SVF: stromal vascular fraction; NPs: low-molecular-weight heparin/protamine nanoparticle.

Fig. 5

Heterotopic SVF cells survived for four weeks and synthesized α-SMA. (a) Representative light micrographs of urethras four weeks after SVF transplantation (donor: LEW-GFP, recipient: wild type-LEW rats) (b) The micrographic images are modified/edited by Adobe Photoshop CS6. Substantial number of GFP positive cells synthesized α-SMA. Scale bar: 100 μm. GFP: green fluorescent protein; α-SMA: alpha-smooth muscle actin.