The use of autologous skeletal muscle derived stem cells in the treatment of induced stress urinary incontinence, an experimental study

Wadie B1, Amer H1, Sherry K1, Gabr M1

Research Type

Basic Science / Translational

Abstract Category

Female Stress Urinary Incontinence (SUI)

Abstract 489
Basic Science: Stress Urinary Incontinence and Benign Prostatic Hyperplasia
Scientific Podium Short Oral Session 27
Friday 31st August 2018
09:30 - 09:37
Hall D
Animal Study Incontinence Cell Culture Stem Cells / Tissue Engineering
1. Urology and nephrology center
Presenter
B

Bassem Wadie

Links

Abstract

Hypothesis / aims of study
Treatment of stress incontinence is challenging. In humans, different techniques evolved throughout ages of research and clinical trials. So far, no standard procedure could be considered “gold”. However, Midurethral slings are by far the most world-wide used procedure. MUS are not without risks and are not economic as well. Many trials, both experimental and human attempted to provide an autologous, efficacious and durable tissue-engineered sling.
This is an experimental pre-clinical study, carried out on 10 female mongrel dogs to study the applicability of autologous skeletal muscle derived cells as a seeded Polyglcolic acid (PGA) scaffold slings as a treatment of SUI in canine model
Study design, materials and methods
The study entailed 10 Mongrel dogs. In 4,  Isolation and expansion of muscle-derived stem cells (MDCs): 
      An open deltoid muscle biopsy was performed. The obtained biopsy specimen of approximately 0.2 g was sent to isolate MDC cells for expansion. Cells were isolated via collagenase Type 1A (Sigma-Aldrich, St. louis, MO, USA) digestion. The cells were cultured on laminin-coated tissue culture flasks in SKGM-2 medium (Lonza, Valkersville, MD). The medium was replaced every 3 days. At confluence, cells were passaged and split 1:2 in tissue culture flasks. After 8 weeks of expansion, the obtained (MDCs) were collected and transported for transplantation. 
Characterization of MDCs:
     The morphology of the expanded cells was evaluated using phase-contrast microscopy. The expression of a muscle marker, desmin, was assessed immunohistochemically with monoclonal anti-desmin antibodies (DakoCytomation, Glostrub, Denmark). 
Cell seeding on scaffolds:
Neoveil absorbable polyglycolic acid sheet (Gunze Limited, Kyoto, Japan) was used. The sheet was cut into pieces (2x3 cm2 ). The scaffold piece was kept immersed in culture medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin for 24 h. at 37 oC before seeding. The scaffold was coated with matrigel and MDCs were then seeded at a concentration of I million cells per cm2, and after 24 h, the seeding side was flipped and the other side was coated with matrigel followed by seeding a concentration of 1 million cells per cm2. Both cell-seeded sides were fully immersed in medium during seeding process. The cell-seeded scaffold was cultured for further 3 days in fresh medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin at 37 oC and then transferred for transplantation.

Urethral pressure (UPP) measurement was carried out in 8 dogs in which induction of incontinence was performed at baseline and 2 weeks after insertion of the sling, using Gaby machine ( Laborie)
6 F dual lumen catheter and 1 ml/min filling rate were used to assess urethral profile in dogs
Results
Four dogs were considered as cases with PGS sling seeded with MDCs while another 4 had only PGA slings without cell seeding.
The urethra with its surrounding tissue was harvested and dogs were sacrificed 4 weeks after sling insertion and were sent for histopathology. 2 dogs were considered as control, in whom no urethrolysis or insertion of slings were carried out
Cross section were taken from dog urethras at the mid urethral segment where the slings have been inserted.
Staining with Hematoxylin and Eosin was carried out first. Masson Trichrome was used as a special stain to identify the presence of muscle fibre aggregates. Desmin immuno histochemical stain was used fro identification of skeletal muscle fibres.
Interpretation of results
UPP show increase of maximum urethral pressure during static measurement in all dogs with a scaffold inserted. The increase ranged from 5-40 cmH20 (Median 23 cmH20)
Histopathology shows significant periurethral proliferation of skeletal muscles in 4 dogs with cell-seeded scaffold, as demonstrated by IHC stain with Desmin. This was exceeding the normal urethra of control dogs in case of # 1& 2. This was not the case in the 4 dogs that had only PGA scaffold as a sling, as compared to the normal urethral wall in control dogs. Fig 1 shows Masson trichome stain of section from dog as well IHC desmin stain
Concluding message
The use of skeletal muscle –seeded PGA scaffold is a practical technique with preserved integrity of histological differentiation in canine model at short term. It could represent a very goop substitute for autologous slings in humans
Figure 1
Figure 2
Disclosures
<span class="text-strong">Funding</span> Institutional <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> Dogs <span class="text-strong">Ethics Committee</span> Hospital IRB