The Use of Autologous Skeletal Muscle Derived Cells as a Sling in the Treatment of Induced Stress Urinary Incontinence, An Experimental Study in Dogs


 Introduction:This is an experimental pre-clinical study, testing the applicability of autologous skeletal muscle derived cells as a treatment of SUI in canine modelMethods:10 Mongrel dogs included. Skeletal muscle biopsy was harvested in 4. 1 month later, incontinence was induced in 8 dogs through urethrolysis. Muscle biopsy was incubated and expanded for 8 weeks. Muscle derived cells were collected and covered a Polyglycolic acid (PGA) scaffold immersed in culture medium and coated with matrigel to be used as a sling. Placed suburethral in 8 dogs; 4 had cell- seeded and 4 had scaffold only. Urethral pressure (UP) measurement was done at baseline 2 &6 weeks after sling insertion. The urethra was harvested 4 weeks after sling insertion for histopathology.Results:UP shows increase of maximum urethral pressure during static measurement in all dogs with a scaffold inserted. The increase ranged from 5-40 cmH20 Histopathology shows significant periurethral proliferation of skeletal muscles in 4 dogs with cell-seeded scaffold. This was maximum in dogs # 1& 2. This was not the case in the 4 dogs that had sling only.Conclusion: Use of skeletal muscle –seeded PGA scaffold is a practical technique with preserved integrity of histological differentiation in canine model.


Introduction
The treatment of stress incontinence is challenging and different surgical techniques evolved throughout numerous clinical trials in order to e ciently treat this condition. So far, no procedure is considered "gold standard". However, a midurethral sling (MUS) is the most widely used procedure and is considered as the standard of care in women with stress incontinence. Synthetic MUS were used for decades but were not without risks and sometimes could be life-threatening. Many trials, both experimental and human, attempted to provide an autologous, e cacious and durable tissue-engineered sling. We conducted this experimental trial with the purpose of assessing the potential of an autologous cell-seeding of a biodegradable scaffold in treating induced incontinence in canines It evaluates a biodegradable seeded (PGA) scaffold enriched with autologous skeletal muscles without the need for a harvest procedure such as that used during rectus fascia sling, being the prototype of autologous slings.

Materials And Methods
The study comprised 10 Mongrel female dogs. Approval from the local Ethics Committee was obtained (Research center Ethics panel, Urology & Nephrology center).
All methods were carried out in accordance with relevant regulations and in accordance with ARRIVE guidelines; including housing, anesthesia and surgical procedures in dogs.
Open episiotomy was carried out 2 weeks prior to the study to facilitate the vaginal approach.
Group 1 comprises 4 dogs in which a cell-seeded sling was applied while in group 2, only the PGA sling was applied.

Isolation and expansion of muscle-derived cells (MDCs):
In 4 dogs (group 1), muscle biopsy was harvested from biceps under general anesthesia. Obtained biopsy averaged 0.2 gm. and was incubated in 0.2% collagenase type 1A in Dulbecco's Modi ed Eagle Medium (DMEM) medium for 20 minutes at 37 C. (Sigma-Aldrich®, St. Louis, USA) digestion. Cells were cultured on laminin-coated tissue culture asks in SKGM-2 medium (Lonza®, Valkersville, MD). Medium was replaced every 3 days. At con uence, cells were passaged and split 1:2 in tissue culture asks. After 8 weeks and 4 passages, MDCs were collected for transplantation.
• Cell seeding on scaffolds: Neoveil absorbable PGA sheet (Gunze Limited®, Kyoto, Japan) was used as (2x3 cm) segments. Seeding started by immersion in culture medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin for 24 h. at 37 o C before seeding. The scaffold was coated with matrigel and MDCs were seeded at a concentration of 1 million cells/ cm 2 . Matrigel was being used in our study in order to increase cell adherence to the scaffold. It is one of the promising materials in this regard. After 24 h. the seeding side was ipped and the other side was treated the same way. Both surfaces were fully immersed in medium during seeding process. Seeded scaffold was cultured for further 3 days in the same culture medium • Induction of incontinence: One month after biopsy, incontinence was induced in 8 dogs through midline suprapubic incision and identi cation of the urethra and bladder. Sharp dissection of the urethra all around until disruption of the periurethral sphincter is achieved. This method is similar to that previously described by Rodriguez et al, with the addition of pubourethral ligament disruption.
Two weeks after induction of incontinence, sling was applied through vaginal incision in suburethral position in all 8 dogs: Cell-seeded scaffold in the rst 4 and scaffold -only in the other 4. The sling is xed to the periurethral tissue. Urethral pressure (UP) measurement was done before (baseline) and 2 weeks after insertion of the sling.
The urethra with its surrounding was harvested and dogs were sacri ced 4 weeks after sling insertion. Figure 1 shows the scaffold and steps of surgical procedure The urethra was xed in 10 % buffered formalin and processed in para n blocks, sectioned at 5 µm and stained with hematoxylin and eosin (H&E), methenamine silver (for reticuline bers detection) and Masson trichrome stain (for brosis detection and muscle bundles delineation).
2 dogs were considered as control, in whom no urethrolysis or insertion of slings were carried out. UP was carried out in these dogs before being sacri ced and their urethra was taken as control.
A owchart for the research procedures was supplemented (Supplement) Results UP shows increase of maximum urethral pressure in all dogs with a scaffold inserted. Table 1 demonstrates change in UP over time. Median maximum urethral closure pressure (MUCP) at baseline in the rst group; where the scaffold was seeded with skeletal muscle cells, was 28.5 cmH20 that increased to 67.5 at 2 weeks and 65.5 at 6 weeks. While in group 2 (scaffold -only group), the median MUCP increased from a basal value of 19 to 21 cmH20 and stayed at this level at 6-week. The increase in (MUCP) ranged from 13-60 cmH20 (Median 39 cmH20) in the rst group. In the second one, the increase of MUCP ranged from -3 to 18 (median 5.5 cmH20) Histopathology: In group 1: urethral lumen was slightly dilated in comparison to the control group; mucosal lining is of near normal thickness. Lamina propria is slightly thicker and no evidence of brous tissue deposition as evidenced with Masson trichrome. Reticular bres are the same as in control group. Thick walled veins are mildly increased in number and caliber compared to control group. The overall muscle layer appears as lavender red bundles in Masson trichrome are normal in thickness with some degree of crowding and disorganization. Areas of newly formed muscle bundles with central nuclei were con rmed.
In group 2:The damaged sphincter shows mildly dilated luminal area, with atrophic urothelial lining. The lamina propria thickness is increased with moderate brous tissue deposition in addition to lower reticular ber content and mild in ammatory lymphocytic in ltrate. The vascular component is unremarkable. There is substantial loss of muscle layer with an overall decreased wall thickness.
IHC for anti-desmin shows signi cantly higher sphincter muscle mass in seeded scaffold specimens as compared to the other group. Newly formed muscle bundles are wider and more randomly aligned, compared to normal sphincter. Staining for S100 shows the presence of nerve bers between the regenerated sphincter muscles. Figure 2 and 3 shows histopathological changes in the two groups.

Discussion
Induction of incontinence by urethrolysis with pubourethral ligament injury was our approach of choice. It resulted in disruption of the native support of the urethra and durable loss of urethral resistance. Incontinence was con rmed by lower maximum urethral closure pressure as compared to control dogs, in which no urethrolysis was carried out Other human trials entailed intraurethral injection of muscle-derived stem cells were published, both in adult women and in children. Although results were promising, some reports turned out to be unfounded.
Another research strategy involved making a structured skeletal muscle tissue in vitro, with the potential of making an all-autologous sling. Many studies evolved to reconstruct skeletal muscle tissue. Some focused on developing a self-organizing tissues, without arti cial scaffolds; others preferred seeding cells on a natural or a synthetic biodegradable substrates e.g. collagen matrices We preferred Polyglycolic acid as a biodegradable scaffold as it was used by Saxena et al & where myoblasts derived from neonatal rats, Fisher CDF-F344, were seeded onto Polyglycolic acid meshes and implanted into the omentum of syngeneic adult Fisher CDF-F344 rats with promising results and comprehensive description of the approach. The advantage of the sling approach is that it applies potentially successful technique with an added effect of autologous skeletal muscle bers to the midurethra To our knowledge, no human study involving biodegradable sling seeded with autologous muscle-derived cells was yet reported. Such a study will be of paramount importance, considering that the current treatment options of women with SUI are far from optimum The question we tried to answer is whether muscle-seeded biodegradable scaffold is any different from a plain one? So, we compared absorbable sling to sling seeded with autologous muscle cells.
Cell -seeded sling proved to be more e cacious than plain PGA. Urethral pressure measurement 6 weeks after sling insertion showed that the median increase of urethral closure pressure in the cell-seeded sling was over 40 cm H20 while in the other group where only PGA sling was applied, it was 5.5 cmH20 Histopathological study of harvested urethral segments 4 weeks after insertion of slings proved the persistence of viable skeletal muscle and nerve bres.
This sling design avoids polypropylene-related adverse events. Application of this technique in humans is easy and promising, based on functional and urodynamic outcome.
One of the shortcomings of our study is reliance on the measurement of maximum urethral pressure in female dogs; that might not be very reproducible. This is probably true according to one study; yet, others have adopted the same parameter we used Conclusion A biodegradable PGA sling seeded with autologous myoblasts shows evidence of survival 6 weeks after insertion in dogs. The use of skeletal muscle -seeded PGA scaffold is a practical technique with preserved histological differentiation in canine model at short term. A) Double -faced seeded scaffold in sterile medium. B)Midline vaginal incision with distal 2/3 of the dog urethra completely exposed, c) PGA sling is attached to the periurethral tissue using 5/0 Vicryl, D) Whole urethra is dissected from the surrounding, and harvested A} Diffuse increase in thickness of skeletal muscle bundles with mild disorganization in PGA seeded with MDC using IHC for desmin stain (brown), B} Minimal increase in thickness of skeletal muscle bundles in PGA only using IHC for desmin stain (brown), C} Abundant nerve bers in between muscle bundles in PGA seeded with MDC using IHC for S100 stain (brown), D} Minimal increase in nerve bers in between muscle bundles in PGA only using IHC for S100 stain (brown) Figure 3 A} Venous plexus is increased in number with marked congestion and mild edema in PGA seeded with MDC, D} Venous plexus is near normal in number with mild congestion with mild in ammatory in ltrate in PGA only, B} Silver stain shows near normal amount of reticular bers (black) in PGA seeded with MDC, E} Silver stain shows lower amount of reticular bers (black) in PGA only, C} The lamina propria expressed absent brous tissue (blue) in PGA seeded with MDC using Masson trichrome stain, F} The lamina propria showed diffuse moderate brous tissue (blue) in PGA only using Masson trichrome stain

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Flowchart.docx