Use of a Latex Biomembrane for Bladder Augmentation in a Rabbit model : Biocompatibility , Clinical and Histological Outcomes

Purpose: To investigate histological features and biocompatibility of a latex biomembrane for bladder augmentation using a rabbit model. Material and Methods: After a partial cystectomy, a patch of a non-vulcanized latex biomembrane (2x4 cm) was sewn at 15, 45 and 90 days after surgery and the bladder was removed. The 5-μm preparations obtained from grafted area and normal bladder were stained with hematoxylin-eosin. Immunohistochemical staining was performed with a primary antibody against alpha-actin to assess muscle regeneration. Results: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. Macbladder. A bladder stone was found in one animal (6.6%). On the 90th day, histology revealed continuity of transitional epithelium of host bladder tissue on the patch area. At this time, the muscle layers were well organized in a similar fashion days p < 0.0001, 45 days p < 0.001, and 90 days p < 0.01. The anti alpha-actin immunoexpression peaked at 45 days, when the graft was observed covered by muscle cells. Conclusion: The latex biomembrane is biocompatible and can be used in models for bladder augmentation in rabbits. It promotes epithelium and muscle regeneration without urinary leakage.


INTRODUCTION
The worsening quality of life and the loss of upper urinary tract function are consequences of permanent reduction of bladder capacity and compliance, caused by neurogenic or non-neurogenic disorders.reduction of intravesical pressure, improvement of urinary continence and preservation of upper urinary Basic and Translational Urology Basic and Translational Urology even today, the most used technique for this purpose.Despite good functional results (2), bladder augmentation with intestinal segments presents disadvantages such as the production of mucus, electrolyte imbalbladder stones, spontaneous bladder perforation and carcinogenesis (1)(2)(3).Auto-augmentation and ureterocystoplasty has emerged as an alternative to override the complications caused by enterocystoplasties, but the second, depends on the presence of a nonfunctioning kidney unit (4,5).
As non-biodegradable synthetic material for bladder augmentation, such as silicon, polytetrafluoroethylene and polypropylene, has been unsuccessful, the development of biodegradable scaffolds seems to be more appropriate because it would allow the appropriate time to host tissue regeneration with dissolution before a foreign body reaction (6).In recent years, the porcine small intestinal submucosa (SIS) and the bladder frequently in experimental studies.They act as biodegradable materials that allow the urothelial and smooth muscle regeneration (7,8).However, and require advanced technique preparation.
The natural latex biomembrane is extracted from the Hevea brasiliensis.Previous experimental studies have proved that the latex biomembrane is a biodegradable material with easy preparation and handling, encouraging tissue formation and angiogenesis (9,10).No evidence of toxicity or allergenic reaction was found when the latex biomembrane was used to reconstruct the tympanic membrane in humans (11).However, the allergen potential of vulcanized latex is well established in latex glove users and other highrisk groups of patients with different latex exposure (12).This background prompted our interest in enlarging the bladder using this type of latex graft.The aim of the study was to investigate the clinical and histological features as well as the biocompatibility of a latex biomembrane for bladder augmentation in a rabbit model.

MATERIALS AND METHODS
A total of 15 adult male New Zealand rabbits weighing 3.0-3.4Kg underwent bladder augmentation with the latex biomembrane.This project was approved by the Animal Research Committee of our 15 (group A), 45 (group B) and 90 (group C) days after grafting.As controls, in each rabbit we excised a bladder full thickness fragment distant from the grafted area.

Membrane Preparation
Matrix preparation followed the technique from the Hevea brasiliensis tree was poured as a thin layer on Petri dishes and dried in an oven at 60ºC for 20 minutes in order to promote the polymerization of latex's constituents.The latex membranes so obtained were immersed in a 0.1% solution of poly-L-lysine hydrobromide (MW 70-140 kD, Sigma) for 24h at room temperature.Then, the membranes were placed into a dry-heat oven at 60ºC for 2 hours, packed and sterilized in ethylene oxide.

Surgical Technique
The animals were anesthetized by intramuscular injection of ketamine (35 mg/kg) and xylazin (5 mg/kg).A partial cystectomy (4.0 cm 2 -2.0 x 2.0 cm), corresponding to nearly 15% of bladder size, was performed through a median laparotomy.A patch of 2.0 x 2.0 cm of the latex biomembrane was grafted onto the remaining host bladder with a continuous suture of 5/0 monofilament absorbable polydioxanone sulfate.Four marking stitches of 5/0 polypropylene were placed outside the bladder wall near the corners of the patch.Perivesical fat was fixed over the bladder wall to cover the graft.

Follow-up
neous to all animals daily for 2 postoperative days.The clinical condition of all animals was evaluated was carried out with an intravenous injection of pentobarbital (60 mg/Kg).A macroscopic inspection was then performed before bladder removal.A silk ligature was placed around the urethra, samples for immunohistochemistry were taken and the bladder in the same solution.

Histology and Immunohistochemistry
and control areas of the bladder were embedded in hematoxylin and eosin in order to evaluate the amount For immunohistochemistry, samples from 24h in ice-cold 0.1 M PBS, ph 7.4, containing 4% paraformaldehyde, followed for cryoprotection in 15% of sucrose for 4h and 30% sucrose overnight were incubated with 3% H2O2 and Pierce solution to block endogenous peroxidase and biotin, respectively.Sections were subsequently incubated with primary antibodies against anti-smooth muscle -vocastra®) and with biotin-conjugated secondary Inc., Burlingame, USA) and streptavidin-conju-ratories Inc.).Color was developed by the addition of DAB (Sigma Chemical, St. Louis, USA).To evaluate the background reaction, procedures were also performed in sections incubated only with the secondary antibodies (indirect technique) or in the absence of antibodies (direct technique).The number of cell with positive staining for alphaactin was estimated by using a camera (Axio Cam, In histological / immunohistochemical evaluation, 10 microscope fields were examined.Cells were counted at a 400X magnification by two different pathologists in a blind manner.The semi-quantitative analysis was determined and expressed as a percentage of inflammatory cells and cells labeled with anti-smooth muscle alpha-> 75%.

Statistical Analysis
Data are provided as medians and range.The comparisons between the treated and controls areas were analyzed by the Wilcoxon test.The relationships between groups were analyzed with analysis of variance (Kruskal-Wallis test), followed by the Dunn's test to compare individual pairings.Statistical analysis was performed using CA, USA) and p values < 0.05 were considered statistically significant.

Macroscopic Evaluation
All rabbits were able to void spontaneously after the operation and no animal exhibited urinary leakage.All bladders had a spherical shape.
On the 15th postoperative day, blood vessels were visible around the grafted area.After a longitudinal incision of the bladder, the presence of the intact.
On the 45th day after surgery, the graft was almost entirely integrated to the host tissue of the native bladder.Bladder wall was thicker, mainly around the latex membrane.One bladder stone (1.5 x 2.0 cm) was observed in one rabbit (6.6%).
After 90 days from surgery, there was a decrease on bladder wall hypertrophy and the graft was indistinguishable from the normal host bladder at inner and outer surfaces.There was blood vessel reduction around the grafted area (Figure -1).

Microscopic Evaluation
On the 15th postoperative day, the luminal surface of the latex matrix was still uncovered by the urothelium.Fibrovascular reaction was presdays after surgery there was a diffuse epithelial and smooth muscle hyperplasia on the graft.At 90 days, there was a reduction on epithelial and muscle hyperplasia, and the urothelium was similar to the native bladder (Figure -2).At this time, the smooth muscle layers were well organized and in a similar direction in comparison to native bladder muscle layers.
process was higher 15 days after the procedure and decreased gradually from the time of grafting, tending to normalization at 90 days.This occurred despite the areas in all groups (Table-1).
The neutrophils and mononuclear cells were decreased with the time of follow-up being restricted around the remaining latex membrane in group C (Figure -3).

Smooth Muscle Layer Regeneration
started from the border toward the center of the graft.
cell regeneration had began poorly and did not exhibit spatial organization, mainly near the anastomosis area.The expression of alpha-actin positive cells peaked C), the smooth muscle cells were well developed the junction between the graft area and host bladder muscle (Figure -4).
The expression of the alpha-actin varied sig-(Kruskal-Wallis' test).Higher expression was detected in grafted area in animals from group B (45th day) that was statistically different in comparison with groups higher in groups B and C and was similar in group A.
(Wilcoxon paired test).There was no change in the expression of alpha-actin in non-grafted areas with time (Table-1).

COMMENTS
The ideal material for bladder augmentation should allow the progressive growth of all components of the normal bladder wall, preserving their mechanical and functional properties (7,8,13).Probst et al. (8) showed that bladder augmentation in rats (32%) due to urinary leakage and/or bladder neck obstruction.However, in the surviving rats, the graft muscle cells of the host and the mucosal lining was complete within 10 days.The ingrowth was complete after 8 weeks, except for neural regeneration, which was only partial.At 12 weeks, the bladder wall muscle structure in the graft was so well developed that it was pears to serve as a framework of collagen and elastin for the ingrowths of all bladder wall components.The high mortality rate caused by urinary leakage in the has also been reported elsewhere (13).Ayyildiz et al. (7) reported that SIS seems to be a viable alternative to the use of intestine in bladder augmentation in rabbits since at the end of 12 months, the long-term

A B A B C
histological features of bladder augmentation was indistinguishable from original bladder.The rabbit model is suitable for bladder tissue engineering studies, but blad-der augmentation with SIS or collagen-based biomatrix exhibits a high rate (37%) of stone formation and/or encrustation of foreign body material (14).

A B C
biomembrane used for bladder augmentation in rabbits allows a progressive ingrowth of all components of the normal bladder wall without postoperative urinary leakage and a low rate of stone formation.After 3 months, the smooth muscle cells were well the junction between the grafted area and host bladder muscle.It also seems that this biodegradable material offers the advantage to reduce the rate of stone formation or encrustation (6.6%) in comparison with SIS, collagen-based biomatrix and bowel segments.The inflammatory process in bladder augmentation with collagen-based biomatrix may be secondary to early congestion of the grafts as well as to stimuli caused by the graft components (15).This reaction was stronger in the first 2 weeks following the surgery, but thereafter its intensity reduces progressively.The inflammatory process must be temporary and not lead to graft rejection (8).The vigorous inflammatory response caused by grafted extracellular matrix is restrict to a TH2 lymphocytes immune response, which results in tissue remodeling rather than tissue destruction or rejection (16).Our data with latex biomembrane showed that the inflammatory reaction was more intense on the 15th postoperative day and decreased significantly later on, which may be regarded as an evidence of no rejection.Previous reports on production of inflammation cytokines induced by latex biomembrane in vitro showed enhancement of production of interleukin-10 and reduction of interferon Y, which suggest that such material elicits an immune response restricted to TH2 lymphocytes (17).It is relevant to stress that during latex biomembrane preparation, one must preserve the native conformation of latex proteins by avoiding temperatures higher than 60ºC and hence the latex vulcanization, otherwise the inflammatory response changes from a tissue repair type toward a rejection type, as shown previously by electron microscopy (9,18).
The exact origin of smooth muscle cells (SMC) in the bladder grafts remains unclear.Some authors suggest a major role for pluripotential stem cells in the graft cellular regeneration (19,20).On the other hand, other authors have demonstrated that muscle layer regeneration occurs by migration of dedifferentiated bladder SMC from the matrixbladder junction (15).Previous experimental studies with the latex biomembrane have shown that it allows the ingrowths of epithelial, submucosal glands and muscular cells of canine esophagus (9).The results of our study suggest that the muscle layer regeneration occurs from the host bladder since alpha-actin was first observed in this area.Bladder augmentation with other extracellular matrix graft showed that the expression of alpha-actin was first visualized on day 4 after the procedure, peaked at day 10 and then decreased.Expression increased again gradually after 3 to 4 weeks and progressed for 12 weeks (14).The early peak of alpha-actin expression was interpreted most likely as a consequence of artifacts caused by the higher number of cells in the matrix rather than the real expression of muscle cells in the matrix.Other studies showed a progressive expression of alpha-actin from 15% in the first 2 weeks to 36% in the 12th week, at a time when muscle layer was well developed and oriented (8).The current study demonstrated that the alpha-actin expression was higher 45 days after surgery and decreased thereafter.At the end of the experiment, muscle layer was well organized, similar to the host tissue.These findings support the idea that latex biomembrane can be used as a matrix in experimental studies of bladder augmentation because it is well tolerated and promotes adequate smooth muscle regeneration.

CONCLUSION
This study demonstrates the biocompatibility of the latex biomembrane as a matrix for bladder augmentation in rabbits.The matrix promotes epithelium and muscle regeneration without urinary leakage.More detailed investigations on its functional properties are warranted in the future.

EDITORIAL COMMENT
I have read an interesting paper entitled, "Use of a biocompatibility, clinical and histological outcomes" prepared by Dr. Domingos and colleagues.Bladder tissue of functional bladder wall.Bladder wall regeneration by means of tissue engineering techniques depends on several supply within the host organism.This paper is dedicated to scaffold.The optimal conditions for the proliferation ence on neo-tissue remodeling were shown to be a desired behavior of both transplanted and host cells.A good scafof mentioned above processes.It was proved that even decellularized animal or human scaffolds were not ideal.The deposition of dense connective tissue and scarring are often observed during healing of tissue-engineered bladder wall.The failure of regeneration can be evoked by allo-or xenogenic cellular remnants within the biological scaffold, urine leakage, etc. Poorly compliant bladder is a result of these unwanted events.Bladder regeneration is much more complicated in a disease condition.The most current experimental works are performed on healthy bladders.There is still a need to create a low immunogenic, high adhesive and biocompatible scaffold for bladder tissue engineering.
They show that even latex can be prepared in such way to be atoxic to bladder cells and promoted in vivo regeneration.They presented bladder wall regeneration induced should be emphasized that scaffold has to be a biological niche for differentiated cells and their stem (undifferentiated) counterparts during in vivo regeneration.It seems that the role of scaffold during the regeneration process is equal to cellular compartment, so I have found this work important.This paper is a very good "background" for the future experiments with the cell-seeded matrices performed on healthy and disease affected bladders.

Department of Urology Department of Tissue Engineering
Nicolaus Copernicus University Bydgoszcz, Poland E-mail: tomaszdrewa@wp.pl

EDITORIAL COMMENT
Many different materials have been investigated for use as scaffolds in tissue engineering.In bladder augmentation models these materials have included collagen, synthetic polyesters such as polyglycolic acid, bladder acellular matrix grafts, and porcine small intestine submucosa.The authors biomembrane scaffold for bladder augmentation in rabbits.Multiple groups from the University of Sao Paulo have shown that the latex biomembrane allows healing in the esophagus (1), the mastoid and dental alveolar bones (2,3) and ulcerated skin (4).In this manuscript, the authors similarly observed progressive smooth muscle growth at the bladder graft site and concluded that further functional studies of the latex biomembrane in the bladder are warranted.
Several concerns will need to be addressed in order to determine if the latex biomembrane bladder augmentation is applicable to human patients.Prior in vitro studies have shown than latex is more cytotoxic to cultured bladder smooth muscle cells compared to other traditional biomaterials (4).More importantly, a large percentage of patients requiring who have a high risk of latex allergy.Nonetheless,

Figure 1 -
Figure 1 -Macroscopic evaluation of the latex biomembrane.A) Presence of the membrane 15 days after surgery.B) Thick bladder wall at 45 days.C) and D) Incorporation of the latex membrane (interior) and spherical architecture (exterior) after 3 months.

Table 1 -
ation.Data expressed as medians and ranges.