PDF-1215.pdf 410 | Cellular and Molecular Urology Purpose: To investigate the feasibility and safety of using biocompat- Materials and Methods: Plasma-treated electrospun unseeded mats and the second and third ones after 4 months, and then, the graft was ex- amined macroscopically with subsequent morphological and histochemi- cal evaluation. Results: mat was very low. All three implantation models showed the same light microscopic morphology, immunohistochemistry, and scanning electron - vorable clinical results. Conclusion: could be a suitable material for the bladder tissue engineering; however, it deserves further investigations. Keywords: urinary bladder, tissue engineering, biocompatible materials, polymers Nasser Shakhssalim,1 Mohammad Mehdi Dehghan,2 Reza Moghadasali,3 Moham- mad Hossein Soltani,4 Iman Shabani,5 Masoud Soleimani6 Bladder Tissue Engineering Using Biocompatible Nanofibrous Electro- spun Constructs Feasibility and Safety Investigation Corresponding Author: Masoud Soleimani, PhD UNRC, No.103, 9th Boustan St., Pasdaran Ave., Tehran, Iran Tel: +98 21 2256 7222 Fax: +98 21 2256 7282 E-mail: msoleimani94@yahoo.com Received November 2011 Accepted December 2011 1Urology and Nephrology Research Center (UNRC), Shahid Labbafinejad Medical Center, Shahid Beheshti Uni- versity of Medical Sciences (SBMU), Tehran, Iran 2UNRC, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3UNRC, SBMU, Department of Stem Cells, Royan Institute, Tehran, Iran 4UNRC, Shahid Labbafinejad Medi- cal Center, SBMU, Tehran, Iran 5UNRC, Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center, Tehran, Iran 6UNRC ,Tarbiat Modares University, School of Medical Science, Hematol- ogy Department and Stem Cell Technology Research Center, Tehran, Iran Cellular and Molecular Urology 411Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L INTRODUCTION Bladder is a unique organ in possessing includes three layers, namely mucosa, submocusa, and detrusor muscle. Detrusor con- sists of smooth muscle cells that have capability communication with sympathic, parasympathic, and somatic nerves make the bladder a challeng- ing organ to be substituted. The bladder is susceptible to a variety of possible abnormalities, other tissue damages or losses, - tion, highlight the need for tissue reconstruction. Bladder reconstruction is done using a segment of the gastrointestinal tract as an alternative ma- trix for the bladder augmentation or replacement. (1) Reconstruction of the bladder with autologous non-urologic tissues does not provide the entire function. Furthermore, there are some important complications that limit the use of these natural matrices, such as metabolic abnormalities, perfo- ration, malignancy, and infection.(2) To overcome the problems related to the using of the intestinal segments for the urinary tract re- construction, many efforts have been made using several biological and synthetic materials, such as the de-epithelialized intestinal segments, sero- muscular intestinal segments, dura mater, perito- neum, and fascia, which have resulted in varying degrees of tissue reconstruction.(3) The use of nat- urally-derived agents, including lyophilized dura and submocusa of porcine small intestine and pla- centa could result in contraction for an unknown period of time.(4) Recently, attention has been turned toward au- to-augmentation and ureterocystoplasty. Auto- augmentation has had disappointing outcomes in long-term follow-up and ureterocystoplasty requires severely dilated ureters. De-epitheliali- zation of the bowel segment may lead to growth of mucosal layer; however, shrinkage of graft and re-epithelialization of afore-mentioned bowel segment are the main limitations.(5) proper native bladder tissue and the distrust in the existence of healthy tissue in the involved bladder reconstruction using native tissue. Previously, biological synthetic materials, such as - - construction, but they had no favorable outcomes. The main reason for their failure was body reac- tion to the foreign agents.(6,7) Permanent synthetic mechanical problems. Recently, some investigators have focused their attention on tissue engineering and using biocom- patible synthetic materials for soft tissue substi- tution. There are two forms of the bladder tissue the isolated and cultured primary cells are seeded on natural or synthetic scaffold and then trans- planted into the host, and subsequently the graft regeneration and maturation could be continued in vivo. The second form is cell independent and the acellular matrix or biodegradable, biocompat- ible scaffolding is transplanted into the host. This acellular matrix is used as a mechanical support - generation. Acellular collagen matrix has many growth factors expected to promote tissue regen- eration,(8) but according to literature, this matrix, seeded or unseeded, has not improved tissue re- generation. Atala and colleagues produced an effective bladder tissue by using cell implanta- Bladder Tissue Engineering | Shakhssalim et al 412 | tion on collagen/polyglycolic acid scaffold. They achieved notable results in compliance, leak point pressure, cellular structure, and phenotypical characteristics in some later cellular transplanta- tion on synthetic scaffold.(11) Synthetic polymers due to their reproducibility in synthesis, also having appropriate mechani- cal properties, are the best candidates for matrix synthesis. Furthermore, it is possible to process The application of synthetic/polyglactin compos- ite may lead to chronic infection, foreign body reaction, implant shrinkage, and rejection of the implant.(12,13) Therefore, it is important to investi- gate the body response to the implanted synthetic unseeded scaffold to check the suitability of us- ing synthetic scaffold for the regeneration of the tissue of interest. Previously, Rohman and asso- ciates reported that cells may show preferential growth on materials displaying mechanical prop- erties that most closely represent those of the de- rived tissue.(14) - - strate. We also evaluated the graft morphological- ly and immunohistochemically after implantation in dogs. MATERIALS AND METHODS Three adult intact female mongrel dogs weighing used in this study. The dogs were determined to be healthy based on the results of physical ex- amination and complete blood count. Animals - - outdoor system during the experiment period. They were fed by a balanced commercial dry maintenance diet (Friskies, Purina, Marne-la-Val- lee, France) once a day, and water was offered ad libitum. The experimental protocol was approved the Netherlands) (2 mg/kg) intramuscularly. This was followed by induction of general anesthesia kg), and maintained by the same drugs. The urinary bladder was approached through a caudal midline abdominal incision, and linear cystotomies of the bladder dome were done. Aug- mentation cystoplasty was performed with the to the third dog. A single layer of continuous in- used for the anastomosis. The omentum was then placed over the graft and secured to the bladder - glycolic acid material before the abdominal inci- sions were routinely closed in three layers (Figure 1). All dogs received teramadol for 3 days and an antibiotic for 14 days. A urethral Foley catheter urine leakage and relieve tension on the suture line. All the animals underwent abdominal ultras- tenography to evaluate urine leakage on the 3rd Cellular and Molecular Urology 413Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L and 7th postoperative days, then monthly after- - gen, and serum level of creatinine were tested twice a week for two weeks, and then monthly after the operation. the second and third ones 4 months later. The graft was investigated macroscopically and then evaluated for light and electronic microscopic morphology as well as immunohistochemistry characteristics. Fabrication and Preparation of PCLL and scaffolds were prepared by double jet electrospin- ning method, as described by Khademhosseini and colleagues and Matthews and associates.(15,16) - and N, N-dimethylformamide (DMF) were pur- chased from Sigma (St. Louis, MO, USA). These materials were used as received without any fur- in chloroform at room temperature and DMF was added to the chloroform just before the electro- spinning process. Many studies have shown that DMF helps during the electrospinning and the (17,18) Experiments were conducted at chloroform to DMF ratio of - tained at 12 wt%. Polylactic acid was dissolved at a concentration of 4 wt% in a solvent mixture of chloroform and DMF. A variable voltage power supply was used for to stock each of the prepared solutions. The elec- trospinning setup utilized in this study consisted of three syringes, a ground electrode (stainless steel drum, with outer diameters of 3 and 5 mm, - - syringe nozzle through PE extension tubing. The needle tip could move in restricted distance along the direction of the deposition area; thus, gave the capability of having a uniform mat. A volt- to the solution and the jet emerging from the nee- dle to the drum collector. The collecting surface consisted of a cylindrical stainless steel collector 1 minute, while for in-vitro assessment, in-vitro the scaffold was produced in about 2 hours. The samples were then washed 3 or 4 times in ster- hydrophilicity, oxygen plasma treatment was per- formed. The bare materials were exposed to oxy- gen plasma at 13.6 MHz for 5 minutes using a Figure 1. A single layer of continuous interlocking sutures with 4-0 polyglycolic acid was used for the anastomosis of PCL/PLLA to the bladder of the third dog. Bladder Tissue Engineering | Shakhssalim et al 414 | diener electronic plasma device. Scanning elec- tron microscopy (SEM) observation morphology was ob-© Tescan, served with scanning electron USA) after sputter coating with platinum. The diameter and the distribution of the diameter were measured using image analyzing software. Nonwoven fabric samples with proliferated cells phosphate-buffered saline (PBS). After rinsing and dehydrating in sequentially increasing etha- sputter coating with platinum. Anterior bladder wall incision was made, and the cutting margin was sewn to the square patch of - For immunostaining, the samples were washed - These cells were permeabilized and blocked in - - tively. Thereafter, the samples were incubated - - tibodies used in this study were Desmin (Santa sc-15367), and alpha-smooth muscle Actin (Sig- ma, A5228). At the end of the incubation time, - - - pus, Japan). Furthermore, for histological exami- nation, the specimens were washed twice with through a series of graded alcohol solutions and - ness. The sections were attached to poly-l lysine for 12 hours, dewaxed in xylene, and stained with Hematoxylin and Eosin. - hours at room temperature, and then, dehydrat- ed through a series of graded alcohol solutions. Once dried, the samples were mounted on alu- minum stubs, sputter-coated with gold-palladium (AuPd), and viewed by using SEM (Tescan VE- RESULTS As demonstrated, this polymer had high inter- action with cells. Hematoxylin and Eosin stain- - dles and urothelial layer were formed. Macroscopic view of the graft site in the bladder - - Cellular and Molecular Urology 415Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L low-up period upto 4 months for the second dog. Hematoxylin and Eosin staining of harvested implantation, showed proper muscle and urothe- (Figures 2A and 2B). Again encrustation was ob- served in intraluminal view of the bladder (Figure immunohistochemical, and SEM results, macro- scopic view of the bladder in the later case after 4 months revealed normal mucosal appearance, and encrustation could not be detected (Figures 3A and 3B). Therefore, it seems that in our ex- - cal results. DISCUSSION The introduction of alternative tissue regeneration technique by using unseeded synthetic polymers has caused enormous controversy. Pattison and colleagues demonstrated that the possible etiolo- gy of limitations of this experience, including in- fection, toxicity, and biocompatibility problems, was related to the micron size of these polymers; thus, these particles could not regenerate the new tissue from the surrounded native cells.(19) study by Vance and associates demonstrated that - Figure 2A. Hematoxylin and Eosin staining of the harvested graft site of the second dog after 4 months of PCL implantation. Figure 2C. Encrustation was observed in intraluminal view of the bladder after implantation of PCL. Figure 2B. The immunohistochemical appearance of the harvested graft site of the second dog after 4 months of PCL implantation. Bladder Tissue Engineering | Shakhssalim et al 416 | - yglycolic acid (PLGA) polymers prepared with - cells growth; - ymers to nano dimensions gained more attention. - completely different cellular response. Thapa and coworkers presented the in-vitro study that revealed the enhancement of proliferation and adhesion of the bladder smooth muscle cells on nano-structural they showed that cell growth increases over the ex- tended periods of 1, 3, and 5 days after using those nano-structural polymers.(21) Another in-vitro study revealed that cellular adhesion on nano-dimen- - icantly in comparison with traditional micron size polymers.(22) Pattison and colleagues produced a nano-rough surface of three dimensional PLGA in the laboratory environment successfully, and then, cellular growth, adhesion, and protein production improved with these particles in comparison to the micron polymers. They recommended that using nano-rough surface of three dimensional PLGA scaffolds may be a proper medium for regenera- tion of the bladder wall cells in in-vivo environ- ment.(23) Harrington and associates reported their in-vitro experience of implantation of multipotent stem cells on nano-dimensional structure(24) and the other similar studies explored the interesting aspects of using these polymers with two different approaches, including bottom-up (self-assembling cells)(25) and top-down (differentiation was solely controlled by nanotexture size).(26) Allogenic matrix with seeded cells used in the canine model brought about more noticeable re- sults than the acellular matrix(27) Figure 3A. Hematoxylin and Eosin staining of the harvested graft site of the third dog after 4 months of PCL/PLLA implantation. Cellular and Molecular Urology 417Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L improvement of regeneration of the bladder wall cells was observed on scaffold with seeded cells functional capacity).(28) Domingos and coworkers revealed that the latex biomembrane as a biocom- patible agent can be used in a rabbit model for the bladder augmentation successfully and this mate- rial promoted epithelium and muscle regeneration with proper clinical and histological outcomes. The transitional epithelium continuity of the host bladder tissue on the patch area and the well- organized muscle layers were detected on the 9th day.(29) The other study demonstrated successful bladder wall grafting in 16 rats. Two months after grafting, proper epithelialization and growth of smooth muscle cells were detected. - - mensions in our canine model (an in-vivo study) as a bioavailable substrate layer in reconstruction of the bladder and regeneration of the epithelium and smooth muscle cells. Further efforts are re- quired in future to evaluate the results of cellular seeding on this nano scaffolds that may revolu- tionize the bladder tissue engineering. CONCLUSION All three implantation models showed the same PLLA model has come up with desirable clinical results. - - folds in nano dimensions in a big animal model for the bladder tissue engineering. However, fur- ther studies by using other nanopolymers may ad- vance the results of tissue engineering in organ reconstruction in the future. Figure 3B. Macroscopic view of the bladder after 4 months showed normal mucosal appearance, and encrustation was not detected. Bladder Tissue Engineering | Shakhssalim et al 418 | CONFLICT OF INTEREST None declared. REFERENCES 1. Scriven SD, Trejdosiewicz LK, Thomas DF, Southgate J. Urothelial cell transplantation using biodegradable syn- thetic scaffolds. J Mater Sci Mater Med. 2001;12:991-6. 2. Kropp BP, Eppley BL, Prevel CD, et al. 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