Bladder Patch Repair Using Mesothelial Cell-Seeded Autologous Granulation Tissue: An Experimental Study in Male Rabbits

Bladder Patch Repair Using Mesothelial Cell-Seeded Autologous Granulation Tissue: An Experimental Study in Male Rabbits | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bladder Patch Repair Using Mesothelial Cell-Seeded Autologous Granulation Tissue: An Experimental Study in Male Rabbits Lin Yang, Zhao Liu, Lei Yan, Yapeng Sui, Laiyuan Qiu, Yi Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5736210/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2025 Read the published version in World Journal of Urology → Version 1 posted 11 You are reading this latest preprint version Abstract Objective Currently, bladder reconstruction using gastrointestinal segments is considered as the gold standard for bladder repair or augmentation. However, postoperative complications including bowel dysfunction, metabolic abnormalities, chronic urinary tract infection and stone formation are common. This study aims to evaluate the utility of the compound graft for bladder patch repair by seeding mesothelial cells onto autogenous granulation tissue. Methods 22 Fr silastic tubes were implanted subcutaneously in 18 male rabbits. The animals were equally divided into two groups. In the experimental group, nine animals underwent omentum biopsies and mesothelial cells were cultured and expanded in vitro. Two weeks after the implantation, the autologous granulation tissue encapsulating the silastic tubes were harvested. The tissue was cut longitudinally and trimmed to 2×2 cm 2 . In the experimental group, mesothelial cells were seeded onto the outer surface of the tissue and cocultured for 7 days to construct a compound graft. In each animal, a full-thickness defect of 2×2 cm 2 in the anterior wall of the bladder was created. The defect was repaired with the constructed compound graft in the experimental group and with the granulation tissue directly in the control group. The bladder was harvested and analyzed grossly and histologically at 1, 2 and 6 months postoperatively. In addition, urodynamics were performed 6 months postoperatively to evaluate the function of the bladder. Results In the experimental group, no severe fibrosis and shrinkage were observed postoperatively and the mucosa at the grafts site appeared sleek and normal in color. Histologically, multilayers of urothelium surrounded by increasingly organized smooth muscles were observed. In contrast, severe contracture and fibrosis of the grafts occured in the control group. Gross examination of bladder mucosa revealed ulcer and stone formation at the site of the grafts. Histologically, accumulation of inflammatory cells and fibroblasts, and extensive scarring occurred. Six months after surgery, the urodynamic results showed that the experimental group had a larger bladder capacity, a lower maximum intravesical pressure and a better bladder compliance. Conclusions Mesothelial cell-seeded granulation tissue can be successfully used for bladder patch repair in male rabbits. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Congenital and acquired defects, such as bladder exstrophy, myelomeningocele, spinal cord injury, and bladder outlet obstruction, can cause decreased bladder capacity, abnormal contractility, and poor compliance, and ultimately lead to poor bladder storage function, incontinence, infection, vesicoureteric reflux, and renal injury[ 1 ]. Currently, bladder reconstruction using gastrointestinal segments is considered as the gold standard for management of these end-stage bladders. However, isolation of these segments from the gastrointestinal tract may cause complications, such as intestinal obstruction and intesitinal fistula. Moreover, secretion of mucus into the urine can cause infection and urinary stone formation[ 2 ]. And metabolic abnormalities and electrolyte disorders may occur due to the absorption function of intestinal mucosa [ 3 , 4 ]. Tissue engineering has been considered as a promising alternative approach for bladder substitution[ 5 ]. Scaffolds seeded with urothelial cells and smooth muscle cells have been reported to be successfully used for bladder reconstruction in experimental studies[ 6 , 7 ]. However, there are some drawbacks to using urothelial cells as seed cells. Firstly, they have a slow rate of in vitro proliferation with low success rate, which could affect the efficiency of tissue construction. Secondly, for patients with urothelial carcinomas, urothelial cells are not the optimal choice of seed cells. Our previous studies have shown that mesothelial cells (MCs) can be used successfully for urethral reconstruction as an alternative for urothelial cells[ 8 , 9 ]. Compared to urothelial cells, MCs are much easier to culture and expand in vitro. Moreover, they can attached onto the scaffold and proliferated to form a mesothelial layer. In addition to seed cells, the choice of scaffold is another key element for the successful tissue repair. Previous studies have long found that implantation of foreign bodies into subcutaneous tissue of animals can induce the formation of an autologous granulation tissue capasule, mainly composed of myofibroblasts and collagens[ 10 ]. This granulation tissue has satisfactory mechanical properties and is entirely self-derived with no immunogenicity. Moreover, it is easy to harvest, avoiding the associated complications. In our previous study, we had constructed a tissue-engeneered tubular graft by seeding MCs onto the granulation tissue and successfully used it for full-thickness urethral repair[ 8 ]. Compared to the urethra, the bladder has a longer contact time with urine. In the present study, we want to investigate whether MCs-granulation tissue compound grafts could be successully used for replacement of bladder tissue. Methods Subcutaneous Tubing Implantation and Harvest All animal experiments were reviewed and authorized by Animal Care and Use Committee of Shandong University (Shandong, China). The study included 18 male New Zealand white rabbits with weight 2–4 kg (mean 2.6 kg) aged from 5 to 6 months. A small midline incision was made in lower abdomen. Subcutaneous tissue bilateral to the incision was dissected from the abdominal wall muscles using surgical scissors and enough space was created for mold implantation. 22 Fr silastic tubes were then implanted inside the subcutaneous tissue to induce the formation of guanulation tissue (Fig. 1 A). One end of each tube was fixed onto the dermis with 2/0 silk thread. A total of four 3-cm-long tubes were implanted for each animal. The skin incision was closed with 3/0 polyglactin 910 (Vicryl). After the surgery, penicillin G sodium (10 5 U/day) was injected intramuscularly for 3 days to prevent infection. Three weeks after implantation, the animals were re-anesthetized and the implants were extracted via the original incision. The tissue wrapping the tubes was harvested for construction of the compound grafts or for bladder reconstruction as a direct control (Fig. 1 B). Harvesting, Culture, and Identification of MCs Omentum biopsies were performed simultaneously with mold implantation in 9 of the 18 animals. The perotoneal cavity was opened and a 2×2 cm 2 specimen of omentum was harvested. As described in our previous study[ 8 ], the fat tissue and blood vessels were eliminated from the tissue using ophthalmic scissors as much as possible. The remaining tissues were minced into small pieces and digested using 0.125% trypsin/0.01% EDTA (Gibco,Grand Island, NY) under continuous agitation at 37℃for 15 mins to dissociate MCs. The digestive solution was filtered through a 150-mesh sterile stainless steel filter and cengrifuged to collect MCs. The isolated MCs were then cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) plus 20% fetal bovine serum (FBS) in a 25-mL culture plate. The cells were expanded to a density of 2×10 6 cells/cm 2 before being seeded. Immunofluorescent staining with antibodies of pancytokeratin AE1/AE3 (Thermo Fisher Scientific, Waltham, USA) and vimentin (Abcam, Cambridge, UK) was used for phenotypic analysis of MCs. Construction of the Compound Graft Upon the third passage, the MCs were dissociated with 0.25% trypsin/0.02% EDTA, centrifuged at 1500 rpm for 5 minutes and resuspended in fresh low-glucose DMEM. The silastic tube was removed from the newly formed tubular tissue and discarded. The tubular tissue was washed three times in phosphate buffered saline (PBS), longitudinally cut and trimmed to a size of 2×2 cm 2 . The tissue was maintained in DMEM for 24 hours to ensure that no bacterial contamination occured. The collected MCs were then homogeneously seeded onto the outer surface of the tissue at a density of 2×10 6 cells/cm 2 . The compound grafts were set static for 4 hours to allow MCs to settle and adhere to the tissue. An adequate amount of DMEM medium was then added to incubate the compound graft at air-liquid level for 7 days. The culture medium was refreshed every 2 days to provide necessary nutrients for cell growth. Samples of the seeded grafts were randomly collected for histological and transmission electron microscopical analysis. As a control, the unseeded tissue was maintained in DMEM for 24 hours. Bladder Surgery and Postoperative Evaluation. The peritoneal cavity was entered through midline lower abdominal incision. The bladder was exposed. 30 ml of normal saline was injected through an 8 F urethral catheter to fill the bladder and a 2×2 cm 2 defect was created in the anterior wall (Fig. 1 C, D). The grafts were then used to repair the defect with a single layer of continuous 4 − 0 polyglycate sutures (Fig. 1 E,F). Four non-absorbable sutures using 4/0 Prolene were left on the 4 corners to mark the edges of the graft. The animals were divided into two groups: nine (the experimental group) received MCs-seeded compound grafts with the mesothelial layer facing the cavity, while the other nine (the control group) received unseeded grafts. The abdominal incision was closed, and a 8 Fr urethral catheter was maintained for 14 days postoperatively to drain the urine and empty the baldder. Cervical collars were used to prevent the animal from removing the urethral catheter. Postoperatively, penicillin G sodium was injected intramuscularly at the dose of 10 5 units per day for 5 days to prevent infection. For each group, three animals were euthanized at 1, 2 and 6 months postoperatively. The peritoneal cavity was reopened and the bladder was exposed for in-situ observation. 30 ml of normal saline was injected into the bladder to observed the morphology of the bladder and ensure whether bladder diverticulum and urinary fistula had formed. In addition, the marking sutures were identified to observe for graft contraction. Then, the entire bladder was harvested and cut accross the posterior wall from the neck to the dome to observe the mucosal morphology of the repair area. Histological Analysis of the Compound Grafts and Retrieved Bladder The granulation tissue and the compound grafts were fixed with 10% buffered formalin and subsequently embedded in paraffin respectively. The serial sections of the tissues were stained with H&E and underwent immunohistochemistry analysis. The antibodies against pan-cytokeratin AE1/AE3 and vimentin were used to detect MCs, while the alpha-smooth muscle actin (α-SMA) antibody (Thermo Fisher Scientific, Waltham, USA) was used to identify myofibroblasts. To dynamically evaluate the neobladder regeneration process, the retrieved bladders at each time point were prepared for H&E staining and immunohistochemistry analysis, respectively, highlighting the margin between the graft and native bladder. The pan-cytokeratin AE1/AE3 antibody was used to identify the urothelium, while the α-SMA antibody was used to identify smooth muscle bundles. The immunolabeling was conducted using the avidin-biotin detection system, and the sections were counterstained with hematoxylin. Transmission Electron Microscopy of the Compound Grafts The seeded grafts were trimmed to a size of 1 mm × 1 mm × 3 mm, fixed immediately with 3% glutaraldehyde, and then postfixed with 1% osmium tetraoxide. The specimen was then stained en bloc with 0.5% uranyl acetate and embedded in Epon 812 for sectioning. Thin sections were subsequently stained and examined using a JEOL-1200EX microscope (JEOL, Japan). Urodynamics Urodynamics were performed without anesthesia at 6 months postoperatively with a urodynamic system (PowerLab 4/26, Adinstruments, Australia). Each group consists of three animals. Another three animals that have not undergone bladder reconstruction surgery were selected as controls. The urethral pressure measurement tube was inserted into the bladder through urethra and the rectal pressure measurement tube was inserted into the rectum. Both tubes were then connected to the urodynamic system. Normal saline was injected into the bladder at a rate of 20 ml/min through an inflow pump. The intravesical pressure (Pves), the abdominal pressure (Pabd) and bladder capacity were monitored simultaneously. Leak point pressure(LPP) and the maximum volume of the bladder(Vol max ) are defined as the Pves and the bladder capacity when the first fluid leakage was observed, respectively. Bladder compliance is defined as the ratio of Vol max to the difference between LPP and Pabd. All values are presented as mean ± SD. Student’s t-test was applied for statistical analysis. P < 0.05 was considered to be statistically significant. Results Three weeks after implantation, all tubes were fully encapsulated with translucent granulation tissue (Fig. 1 B). Histological analysis confirmed that the tissue consisted of a wall of myofibroblasts which stained positively for α-SM actin and vimentin, and a collagen matrix (Fig. 2 ). Transmission electron micrograph showed that the myofibroblasts were spindleshaped and contained large amounts of synthetic organelles (Fig. 3 ). With the aforementioned procedure, MCs were successfully cultured in vitro and maintained their epithelial cell morphology of characteristic "cobblestone-like" growth pattern at the third passage (Fig. 4 A). Immunochemistry staining confirmed the positive expression of both AE1/AE3 and vimentin in MCs (Fig. 4 B,C). The collected MCs could be easily seeded onto and attached to the surface of the autologous granulation tissue. Histological analysis of the MCs-seeded compound graft showed a single layer of MCs forming on the outer surface of the tissue after incubation for 7 days (Fig. 2 D,E,F). The transmission electron microscopy confirmed the presence of characteristic surface microvilli and tight junctions between MCs, indicating their functional integrity (Fig. 3 ). All animals survived the surgery. Prior to harvesting the bladder, 30 ml of normal saline was injected into the bladder for observation of the morphology and size of the repaired area. No diverticulum and urine leakage occurred in all animals. In the control group, severe adhesion was observed between the repaired area and surrounding tissues by one month after surgery. The texture of the repaired area was relatively hard (Fig. 5 A). Remarkable contracture and scarring of the grafts was observed at 2 and 6 months after implantation (Fig. 5 D,E). Gross examination of the bladder mucosa by 1 month revealed ulcer, necotic tissue and stone formation at the repair area with no bladder mucosa regenerated on the surface of the graft (Fig. 5 B). By 2 months, the ulcer area decreased in size and the bladder mucosa had partially covered the repair area (Fig. 5 D). Stone could still be observed. Six months after surgery, the ulcer and stone disappeared and were completely replaced by pale and stiff scar tissue (Fig. 5 F). In contrast, in the experimental goup, no severe fibrosis and shrinkage were observed in the bladder filling state as indicated by the marking sutures (Fig. 6 A,C,E). The mucosa at the grafts site appeared sleek and normal in color with no ulcer and stone formation at different time points postoperatively (Fig. 6 B,D,F). By 1 and 2 months postoperatively, the thickness of the bladder wall at the graft site was thinner than that of normal bladder. Six months postoperatively, the wall thickness of the graft increased and became similar to normal bladder. The bladder repair progress were further evaluated by histological analysis. In the control group, H&E staining and immunohistochemical analysis by 1 month revealed necrotic tissue on the surface of the grafts with no epithelial layers formed. Large amounts of inflammatory cells accumulated beneath the necrotic tissue. Fibroblasts were observed within the grafts especially in the margin with native bladder tissue. No smooth muscle bundles and capillaries were observed within the grafts (Fig. 7 A,B,C). By 2 months, the necrotic tissue nearly disappeared, but inflammatory cells were still observed on the surface of the grafts. Urothelium began to grow into the grafts from the margin, but no complete urothelial layers had been formed. Fibroblasts subsided significantly and fibrous tissues were found within the grafts. Only a small amount of smooth muscle bundles can be seen only at the edge of the graft, although newly formed capillaries were observed within the graft (Fig. 7 D,E,F). By 6 months, although inflammatory cells subsided and the urothelium was well developed, only scant and disordered smooth muscle bundles were observed within the grafts (Fig. 7 G,H,I). In the experimental group, histological analysis by 1 month showed that the grafts were fully covered by multilayered continuous epithelial layers (Fig. 8 A,B,C). The epithelium stained positively with antibody of AE1/AE3, but negatively with antibody of vimentin (data not shown), indicating that the original mesothelium had been completely replaced by the newly formed urothelium. Unlike the unseeded group, no significant infiltration of inflammatory cells into the grafts was observed. Sparse bundles of smooth muscle were observed to infiltrate the grafts at the site of anastomosis (Fig. 8 C). Neovascularization began to form in the submucosal layer (Fig. 8 C). By 2 months, the urothelium became more mature with more mucosal furrows. Meanwile, the density of the newly formed smooth muscle bundles and capillaries increased (Fig. 8 D,E,F). By 6 months, the way that the smooth muscle bundles were arranged became more closely aligned with the inner circular-outer longtidinal arrangement of the normal bladder (Fig. 8 G,H,I). The margin between the graft and native bladder became nearly indistinguishable. In addition to histological analysis, the repair effects were also evaluated by urodynamics before sampling at 6 months post-surgery. The results revealed significant differences between the normal, experimental, and control groups in terms of Vol max , LPP and bladder compliance (Fig. 9 ). The normal group had a Vol max of 86.33 ± 1.53 ml, the experimental group 83.33 ± 2.08 ml, and the control group 76.33 ± 3.22 ml. In terms of LPP, the normal group measured 16.62 ± 1.55 mmHg, the experimental group 18.77 ± 1.79 mmHg, and the control group 24.02 ± 1.22 mmHg. Bladder compliance was highest in the normal group at 6.66 ± 0.71 ml/mmHg, followed by the experimental group at 5.64 ± 0.8 ml/mmHg, and the control group at 3.87 ± 0.13 ml/mmHg. Discussion In the last two decades, tissue engineering has been focusing on bladder tissue reconstruction. Although much progress has been achieved in this field, most studies still remains confined to the stage of experimental research. One of the key elements for successful repair is the appropriate selection of seed cells. From the perspective of the histological structure of the urinary system, urothelial cells and smooth muscle cells are ideal sources of seed cells, and some successful experiences have been gained in animal experiment studies[ 5 ]. However, there are some limitations such as difficulties in in-vitro culture and slow expansion speed of urothelial cells, which makes it difficult to ensure the synchronous expansion of the two types of seed cells in vitro, leading to a longer period and higher failure rate of tissue construction. Moreover, when a patient is accompanied by urothelial tumor, autologous urothelial cells are not suitable for use as seed cells. These limitations have become important factors that impede the clinical applications of tissue engineering for reconstruction of the urinary tract. Therefore, it is necessary to find a substitute source of seed cells that is plentiful, easy to obtain, easy to survive and expand in vitro, and suitable for urinary tract repair. Despite originating from the mesoderm, MCs display epithelial cell growth patterns and express epithelial cell markers[ 11 ]. In patients with peritoneal dialysis, despite continuous exposure to dialysate, there was no significant damage or fibrosis of the peritoneum, indicating the good tolerance of MCs to similar components to urine. Peritoneum has also been attempted as a graft for urethra repair in experimental research, and the results were promising [ 12 , 13 ]. In our previous studies[ 8 , 9 ], we applied MCs as seed cells to repair urethra defect and the seeded MCs successfully prevented graft fibrosis and contracture, demonstrating that MCs could serve as impermeable barriers to harmful urine components. In the present study, we further investigated the feasibility of using MCs for bladder reconstruction. Although the bladder mucosa has a longer contact time with urine as a storage organ, promising results were found. In the control group, we observed a tissue repair process similar to that after transurethral resection of bladder tumors (TURBt), where necrotic tissue or stones first formed on the surface, followed by formation of scar tissue. By contrast, no signs of excessive accumulation of inflammatory cells and fibrosis were observed in the grafts seeded with MCs. We observed that the inflammatory cells accumulated mainly on the surface of the graft in the unseeded group, indicating that urine infiltration was the major cause. Our findings further confirmed that the seeded MCs could form an effective mucosal barrier for bladder repair. Moreover, MCs also possess anti-inflammatory properties. Studies have shown that MCs can effectively inhibit the proliferation of CD4 + and CD8 + T lymphocytes by secreting anti-inflammatory factor TGF-β[ 14 ]. Another in vitro study showed that MCs consume L-arginine through the secretion of arginase I, which subsequently inhibits the activation of CD4 + lymphocytes[ 15 ]. As seed cells, the anti-inflammatory properties of MCs might contribute to reducing the inflammatory response at the repair site, which was beneficial for tissue regeneration and repair. Interestingly, we observed that urothelium in the seeded grafts regenerated much faster than that in the unseeded grafts. It is still unclear how the MCs promoted the regeneration of uroepithelial cells. It has been proved that MCs can be transformed into smooth muscle cells, myofibroblasts, endothelial cells, and even bone and cartilage tissue under specific in-vivo environments, participating in the development or reconstruction of tissue and organs [ 16 – 18 ]. In-vitro studies have also confirmed that MCs can be successfully induced to differentiate into osteoblasts, adipocytes, and smooth muscle cells [ 19 ]. In view of the pluripotent nature of MCs, there is the possibility that MCs underwent transdifferentiation into urothelial cells under the stimulation of urine-based environment. However, it might also be possible that MCs just sloughed off the grafts and were gradually replaced by local urothelium, considering the capability of the urothelium to migrate and proliferate[ 5 ]. The basement membrane or growth factors secreted by MCs may accelerate the migration of surrounding urothelial cells[ 20 ]. The formation of complete urothelial layers over the grafts, in turn, promoted the regeneration of smooth muscle bundles, as the urothelium is thought to play a major role in the differentiation and maintenance of the bladder muscle layer [ 21 ]. Anyway, the destiny of MCs after implantation and how MCs promoted urothelium regeneration needs to be further investigated. In addition to seed cells, the selection of scaffold materials is also critical to the success of tissue repair. An ideal scaffold material should possess good cell compatibility, mechanical strength, and degradability[ 22 ]. Acellular matrix is commonly used as a scaffold material for urinary tract tissue engineering, including bladder acellular matrix, small intestinal submucosa layer acellular matrix, vascular acellular matrix, and et al[ 5 ]. However, the preparation process is time-consuming, and continuous agitation and enzyme digestion can damage the mechanical properties of the scaffold. Additionally, incomplete acellularization can lead to immune reactivity and induce local inflammatory reactions, which is harmful to tissue repair. In the present study, we implanted silicone tubes into the subcutaneous tissue of animals and induced the formation of an autologous granulation tissue, mainly composed of myofibroblasts and collagen matrix. This tissue is of fully autologous origin, without any immunogenicity. It does not require decellularization processing, and thus its mechanical properties and biological activity can be well preserved. Another feature of this tissue is that it resembles corneal tissue with no vascular growth inside, and that its cells' activity fully relies on the nutrients provided by the surrounding tissue fluid. This suggests that it may have better tolerance to the ischemic environment during the initial period of urinary tract substitution. This has been confirmed in our previous study[ 8 , 23 ], in which we successfully applied this kind of tissue in the repair of long urethral defect. For the tissue engineering construction of muscular organs, in addition to seeding epithelial cells, it is necessary to concurrently or sequentially seed cells that produce extracellular matrix, such as smooth muscle cells, fibroblasts, and mesenchymal stem cells, to improve the mechanical properties and biocompatibility of the graft[ 24 – 26 ]. This autologous granulation tissue is already rich in myofibroblasts, so only epithelial cells need to be seeded, which shortens the preparation period, simplifies the construction process and reduces the cost. Myofibroblasts have a morphology and function similar to smooth muscle cells. In the present study, myofibroblasts were observed to be completely replaced by smooth muscle cells after being used for bladder repair. Previous studies have suggested that myofibroblasts can be converted into smooth muscle cells under stimulation of cyclic mechanical stretch[ 27 ], while others have proposed that myofibroblasts secrete chemotactic factors that induce host smooth muscle cells to migrate into the graft[ 28 ]. One month after surgery, we observed the coexistence of myofibrioblasts and smooth muscle bundles within the seeded grafts. Two months after surgery, myofibroblasts were completely replaced by smooth muscle cells. However, we also observed that newly formed smooth muscle bundles firstly appeared at the edges of the graft and then migrated towards the center over time. Therefore, we speculated that both mechanisms were involved in smooth muscle regeneration. Regardless of the mechanism, our study confirmed that the granulation tissue sucessfully promoted smooth muscle regeneration in the presence of an effective epithelial barrier, which was crucial to maintain the structural and functional integrity of the bladder. This was also reflected by the results of urodynamics, which showed the experimental group had a larger bladder capacity and better bladder compliance. The exact mechanism by which myofibroblasts promote smooth muscle regeneration needs to be fully elucidated in the future study. There were several limitations in this study. First, the bladder defect was created in normal healthy bladder, which could not fully simulate the clinical situation. Second, the number of experimental animals is relatively small. To precisely evaluate the success rate, larger sample sizes are needed. Third, as a small-bodied animal, rabbit may not provide as much information for clinical problems. Large-scale animal research is necessary in the future. Finally, as a preliminary study, we have not addressed the repair of a significant bladder defect. The main aim of the study was to evaluate the ability of the constructed tissue to regenerate bladder tissue. In the future study, we will investigate the effect of the constructed tissue for larger bladder defect repair. In summary, we successfully constructed a kind of compound grafts with an inner mesothelium, and a contractile wall of myofibroblasts and collagens. They could guide bladder regeneration for a defect of 2×2 cm 2 in male rabbits. Considering clinical applications, the human scrotum might be a suitable option for mold implantation, which resembles subcutaneous tissue of rabbits with a lack of fat tissue. It might become a promising technique in the clinical use for bladder reconstruction in human after further investigations. Conclusions Our research has demonstrated that granulation tissue seeded with mesothelial cells can serve as an effective material for bladder patch repair in male rabbits. This approach leverages the unique properties of mesothelial cells, which include their ability to promote tissue regeneration and healing. In studies conducted on male rabbits, the mesothelial cell-seeded granulation tissue was observed to integrate well with the native bladder tissue, facilitating the restoration of bladder functionality and maintaining structural integrity. These findings suggest the potential for this technique to provide a biocompatible and efficient alternative for bladder repair, opening avenues for further exploration in regenerative medicine and potential applications in clinical settings. Declarations Conflict of interest The authors declare no competing interests. Funding This work was supported by the grants from Natural Science Foundation of Shandong Province (ZR2021MH049) Author Contribution L.Y. and Z.L. wrote the manuscript. G.G. planned the study. L.Y., Y.S., L.Q. and Y.S contributed to analysis and discussion of the data. J.Z., J.S., and Q.C. analyzed the results. G.G supervised the project. All authors participated in animal experiments. Acknowledgements Natural Science Foundation of Shandong Province. Data availability No datasets were generated or analysed during the References Horst M, Eberli D, Gobet R, Salemi S (2019) Tissue Engineering in Pediatric Bladder Reconstruction-The Road to Success. 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Cite Share Download PDF Status: Published Journal Publication published 07 May, 2025 Read the published version in World Journal of Urology → Version 1 posted Editorial decision: Revision requested 03 Feb, 2025 Reviews received at journal 27 Jan, 2025 Reviewers agreed at journal 19 Jan, 2025 Reviewers agreed at journal 18 Jan, 2025 Reviewers agreed at journal 14 Jan, 2025 Reviews received at journal 08 Jan, 2025 Reviewers agreed at journal 31 Dec, 2024 Reviewers invited by journal 31 Dec, 2024 Editor assigned by journal 31 Dec, 2024 Submission checks completed at journal 31 Dec, 2024 First submitted to journal 30 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5736210","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":396309841,"identity":"f294355a-e82c-4b81-af46-769a397ac33b","order_by":0,"name":"Lin Yang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Yang","suffix":""},{"id":396309842,"identity":"00fe01cb-ac36-428d-91c5-0d7c22b21cc8","order_by":1,"name":"Zhao Liu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Liu","suffix":""},{"id":396309843,"identity":"cb03107a-fde5-4e2f-915b-69841e02c8be","order_by":2,"name":"Lei Yan","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Yan","suffix":""},{"id":396309844,"identity":"e1496f3a-bb08-4418-933c-9ef60af52814","order_by":3,"name":"Yapeng Sui","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yapeng","middleName":"","lastName":"Sui","suffix":""},{"id":396309845,"identity":"830f6392-1194-4130-b8cd-1a960f07d412","order_by":4,"name":"Laiyuan Qiu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Laiyuan","middleName":"","lastName":"Qiu","suffix":""},{"id":396309846,"identity":"4021fa0b-67ce-44f3-9f2f-7ecc1bfeaa48","order_by":5,"name":"Yi Sun","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Sun","suffix":""},{"id":396309847,"identity":"de97481c-139d-4122-aff7-77e9cdff136f","order_by":6,"name":"Jianguo Zheng","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jianguo","middleName":"","lastName":"Zheng","suffix":""},{"id":396309848,"identity":"bf7f21bd-e90d-47ce-a454-cfa52fcbd946","order_by":7,"name":"Jiajia Sun","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Sun","suffix":""},{"id":396309849,"identity":"6b516baa-ab9e-4de1-bce6-0b00b117f3a6","order_by":8,"name":"Qinzheng Chang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qinzheng","middleName":"","lastName":"Chang","suffix":""},{"id":396309850,"identity":"a57f4992-73c6-416c-b349-2c8864e56b2c","order_by":9,"name":"Gangli Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYFAC5oYDCRU2cmzszQeI1cLY+ODBmTRjPp5jCURraTZ82HYocZ5EjgJxGuT9F7ZJJLAdSG9jyGFg+FGxjbAWwxsPgVp47uS2MZw9wNhz5jYRWmYcBGqReJbbxtiXwMzYRrQWg8PpbMw8BsRpkedvbDZISDicwMZGrBYDCWAgJxxIM2zjYUs4SJRf5PsPHzj485+NvPz8xwcf/KggxpYbCQjOAcLqwbYQp24UjIJRMApGMgAAYZtDA3sHvVcAAAAASUVORK5CYII=","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Gangli","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2024-12-30 14:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5736210/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5736210/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00345-025-05611-7","type":"published","date":"2025-05-07T15:56:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72841501,"identity":"fabb0338-5dc3-4a20-b535-de3bece93f3d","added_by":"auto","created_at":"2025-01-02 18:22:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eA, Silastic tube was implanted inside the subcutaneous tissue. B, Silastic tubes were encapsulated with autologous granulation tissue three weeks after implantation. C, Four suspension stitches were sutured onto the anterior wall of the bladder. D, A 2×2 cm2 bladder defect was created. E, The autologous granulation tissue was trimmed to 2×2 cm2 before transplatation . F, The bladder defect was repaired with the tissue.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/a6f5f2b576d637cc0d9ffbaf.png"},{"id":72841503,"identity":"6eb66a48-f0f6-46e0-bc3d-d8bf86683404","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of the grafts. A, H\u0026amp;E staining of the granulation tissue (400×). B, Immunostaining for cytokeratin AE1/AE3 of the granulation tissue (400×). C, Immunostaining for α-SMA of the granulation tissue (400×). D, Immunostaining for vimentin of the granulation tissue (400×). E, H\u0026amp;E staining of the seeded graft (400×). F, Immunostaining for cytokeratin AE1/AE3 of the seeded graft (400×). G, Immunostaining for α-SMA of the seeded graft (400×). H, Immunostaining for vimentin of the seeded graft (400×).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/4b36f0848f375554559faa3e.png"},{"id":72841502,"identity":"51626b7f-8a76-4f5e-8386-5406930c573f","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron micrographs of the seeded graft. A, MCs lining the graft with characteristic surface microvilli (10000×). B, Tight junction between neighboring MCs indicated by the arrow (30000×). C, Spindleshaped myofibroblasts with large amounts of synthetic organelles (15000×).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/fb6a94aff9b68c18b651ab0b.png"},{"id":72841507,"identity":"696d38ba-7e28-4c66-87da-dde22fe339c6","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1156521,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro cultured MCs. A, Phase contrast photomicrograph of cultured cells at third passage (100x). B, Immunohistochemical staining for cytokeratin AE1/AE3 of MCs (400x). C, Immunohistochemical staining for vimentin of MCs (400x).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/3eb627b6b10d3de3778c7b95.png"},{"id":72841505,"identity":"346ee659-2833-4d7f-985b-cd6cc0aa0251","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eGross examination of the retrieved bladder in the control group. A, Morphology of the bladder at 1 month after surgery. B, Ulcer and stone formation at the site of the graft at 1 months after surgery. C, Morphology of the bladder at 2 months after surgery. D, Ulcer was still observed at 2 months after surgery. E, Morphology of the bladder at 6 months after surgery. F, Scar formaiton at the site of the graft at 6 months after surgery.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/3df8913a1736aca9f3d4aafd.png"},{"id":72841519,"identity":"7a2303a6-c3f3-4d37-8453-dc9b3620d604","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eGross examination of the retrieved bladder in the experimental group. A, Morphology of the bladder at 1 month after surgery. B, Mucosal morphology at the site of the graft at 1 months after surgery. C, Morphology of the bladder at 2 months after surgery. D, Mucosal morphology at 2 months after surgery. E, Morphology of the bladder at 6 months after surgery. F, Mucosal morphology at 6 months after surgery.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/eea51c27ef83fc21c2fc59d7.png"},{"id":72841509,"identity":"3d4ecca7-3c3f-42c4-a799-856e98c2e728","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of the margin between native bladder and the graft in the control group. A, H\u0026amp;E staining at 1 month after surgery. B, Immunostaining for cytokeratin AE1/AE3 at 1 months after surgery. C, Immunostaining for α-SMA at 1 months after surgery. D, H\u0026amp;E staining at 2 month after surgery. E, Immunostaining for cytokeratin AE1/AE3 at 2 months after surgery. F, Immunostaining for α-SMA at 2 months after surgery. G, H\u0026amp;E staining at 6 months after surgery. H, Immunostaining for cytokeratin AE1/AE3 at 6 months after surgery. I, Immunostaining for α-SMA at 6 months after surgery.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/209823f598ca6f1b47f16c16.png"},{"id":72841511,"identity":"2127ee59-4c08-4d42-a113-5e27abe3e811","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of the margin between native bladder and the graft in the experimental group. A, H\u0026amp;E staining at 1 month after surgery. B, Immunostaining for cytokeratin AE1/AE3 at 1 months after surgery. C, Immunostaining for α-SMA at 1 months after surgery with the higher magnification showing the newly formed smooth muscle bundles and capillaries. D, H\u0026amp;E staining at 2 month after surgery. E, Immunostaining for cytokeratin AE1/AE3 at 2 months after surgery. F, Immunostaining for α-SMA at 2 months after surgery with the higher magnification showing the newly formed smooth muscle bundles. G, H\u0026amp;E staining at 6 months after surgery. H, Immunostaining for cytokeratin AE1/AE3 at 6 months after surgery. I, Immunostaining for α-SMA at 6 months after surgery. Arrows indicated the margin.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/4a0830a845182d1c99523eb4.png"},{"id":72841520,"identity":"1832cdab-8031-4988-aab1-944b789087d5","added_by":"auto","created_at":"2025-01-02 18:22:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":374418,"visible":true,"origin":"","legend":"\u003cp\u003eUrodynamic examination at 6 months after surgery. A, Changes in intravesical pressure (Pves) during infusion. Vertical axis: Pves (mmHg). Horizontal axis: infusion time (seconds). B, Changes in abdominal pressure (Pabd) during infusion. Vertical axis: Pves (mmHg). Horizontal axis: infusion time (seconds). C, Bladder capacity, leak point pressure (LPP) and bladder compliance at 6 months after surgery. *P \u0026lt; 0.05; #P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/e050516042c5e6107a8b32d9.png"},{"id":82537381,"identity":"df0a03e1-3cf3-473d-90b6-ead495652327","added_by":"auto","created_at":"2025-05-12 15:58:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2165318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5736210/v1/1406adc7-1100-402e-9e5e-0d8f3e63c037.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bladder Patch Repair Using Mesothelial Cell-Seeded Autologous Granulation Tissue: An Experimental Study in Male Rabbits","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital and acquired defects, such as bladder exstrophy, myelomeningocele, spinal cord injury, and bladder outlet obstruction, can cause decreased bladder capacity, abnormal contractility, and poor compliance, and ultimately lead to poor bladder storage function, incontinence, infection, vesicoureteric reflux, and renal injury[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, bladder reconstruction using gastrointestinal segments is considered as the gold standard for management of these end-stage bladders. However, isolation of these segments from the gastrointestinal tract may cause complications, such as intestinal obstruction and intesitinal fistula. Moreover, secretion of mucus into the urine can cause infection and urinary stone formation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. And metabolic abnormalities and electrolyte disorders may occur due to the absorption function of intestinal mucosa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Tissue engineering has been considered as a promising alternative approach for bladder substitution[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Scaffolds seeded with urothelial cells and smooth muscle cells have been reported to be successfully used for bladder reconstruction in experimental studies[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, there are some drawbacks to using urothelial cells as seed cells. Firstly, they have a slow rate of in vitro proliferation with low success rate, which could affect the efficiency of tissue construction. Secondly, for patients with urothelial carcinomas, urothelial cells are not the optimal choice of seed cells. Our previous studies have shown that mesothelial cells (MCs) can be used successfully for urethral reconstruction as an alternative for urothelial cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared to urothelial cells, MCs are much easier to culture and expand in vitro. Moreover, they can attached onto the scaffold and proliferated to form a mesothelial layer. In addition to seed cells, the choice of scaffold is another key element for the successful tissue repair.\u003c/p\u003e \u003cp\u003ePrevious studies have long found that implantation of foreign bodies into subcutaneous tissue of animals can induce the formation of an autologous granulation tissue capasule, mainly composed of myofibroblasts and collagens[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This granulation tissue has satisfactory mechanical properties and is entirely self-derived with no immunogenicity. Moreover, it is easy to harvest, avoiding the associated complications. In our previous study, we had constructed a tissue-engeneered tubular graft by seeding MCs onto the granulation tissue and successfully used it for full-thickness urethral repair[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Compared to the urethra, the bladder has a longer contact time with urine. In the present study, we want to investigate whether MCs-granulation tissue compound grafts could be successully used for replacement of bladder tissue.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eSubcutaneous Tubing Implantation and Harvest\u003c/p\u003e \u003cp\u003e All animal experiments were reviewed and authorized by Animal Care and Use Committee of Shandong University (Shandong, China). The study included 18 male New Zealand white rabbits with weight 2\u0026ndash;4 kg (mean 2.6 kg) aged from 5 to 6 months. A small midline incision was made in lower abdomen. Subcutaneous tissue bilateral to the incision was dissected from the abdominal wall muscles using surgical scissors and enough space was created for mold implantation. 22 Fr silastic tubes were then implanted inside the subcutaneous tissue to induce the formation of guanulation tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). One end of each tube was fixed onto the dermis with 2/0 silk thread. A total of four 3-cm-long tubes were implanted for each animal. The skin incision was closed with 3/0 polyglactin 910 (Vicryl). After the surgery, penicillin G sodium (10\u003csup\u003e5\u003c/sup\u003e U/day) was injected intramuscularly for 3 days to prevent infection. Three weeks after implantation, the animals were re-anesthetized and the implants were extracted via the original incision. The tissue wrapping the tubes was harvested for construction of the compound grafts or for bladder reconstruction as a direct control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHarvesting, Culture, and Identification of MCs\u003c/p\u003e \u003cp\u003eOmentum biopsies were performed simultaneously with mold implantation in 9 of the 18 animals. The perotoneal cavity was opened and a 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e specimen of omentum was harvested. As described in our previous study[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the fat tissue and blood vessels were eliminated from the tissue using ophthalmic scissors as much as possible. The remaining tissues were minced into small pieces and digested using 0.125% trypsin/0.01% EDTA (Gibco,Grand Island, NY) under continuous agitation at 37℃for 15 mins to dissociate MCs. The digestive solution was filtered through a 150-mesh sterile stainless steel filter and cengrifuged to collect MCs. The isolated MCs were then cultured in low-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) plus 20% fetal bovine serum (FBS) in a 25-mL culture plate. The cells were expanded to a density of 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e before being seeded. Immunofluorescent staining with antibodies of pancytokeratin AE1/AE3 (Thermo Fisher Scientific, Waltham, USA) and vimentin (Abcam, Cambridge, UK) was used for phenotypic analysis of MCs.\u003c/p\u003e \u003cp\u003eConstruction of the Compound Graft\u003c/p\u003e \u003cp\u003eUpon the third passage, the MCs were dissociated with 0.25% trypsin/0.02% EDTA, centrifuged at 1500 rpm for 5 minutes and resuspended in fresh low-glucose DMEM. The silastic tube was removed from the newly formed tubular tissue and discarded. The tubular tissue was washed three times in phosphate buffered saline (PBS), longitudinally cut and trimmed to a size of 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e. The tissue was maintained in DMEM for 24 hours to ensure that no bacterial contamination occured. The collected MCs were then homogeneously seeded onto the outer surface of the tissue at a density of 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e. The compound grafts were set static for 4 hours to allow MCs to settle and adhere to the tissue. An adequate amount of DMEM medium was then added to incubate the compound graft at air-liquid level for 7 days. The culture medium was refreshed every 2 days to provide necessary nutrients for cell growth. Samples of the seeded grafts were randomly collected for histological and transmission electron microscopical analysis. As a control, the unseeded tissue was maintained in DMEM for 24 hours.\u003c/p\u003e \u003cp\u003eBladder Surgery and Postoperative Evaluation.\u003c/p\u003e \u003cp\u003eThe peritoneal cavity was entered through midline lower abdominal incision. The bladder was exposed. 30 ml of normal saline was injected through an 8 F urethral catheter to fill the bladder and a 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e defect was created in the anterior wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). The grafts were then used to repair the defect with a single layer of continuous 4\u0026thinsp;\u0026minus;\u0026thinsp;0 polyglycate sutures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE,F). Four non-absorbable sutures using 4/0 Prolene were left on the 4 corners to mark the edges of the graft. The animals were divided into two groups: nine (the experimental group) received MCs-seeded compound grafts with the mesothelial layer facing the cavity, while the other nine (the control group) received unseeded grafts. The abdominal incision was closed, and a 8 Fr urethral catheter was maintained for 14 days postoperatively to drain the urine and empty the baldder. Cervical collars were used to prevent the animal from removing the urethral catheter. Postoperatively, penicillin G sodium was injected intramuscularly at the dose of 10\u003csup\u003e5\u003c/sup\u003e units per day for 5 days to prevent infection.\u003c/p\u003e \u003cp\u003eFor each group, three animals were euthanized at 1, 2 and 6 months postoperatively. The peritoneal cavity was reopened and the bladder was exposed for in-situ observation. 30 ml of normal saline was injected into the bladder to observed the morphology of the bladder and ensure whether bladder diverticulum and urinary fistula had formed. In addition, the marking sutures were identified to observe for graft contraction. Then, the entire bladder was harvested and cut accross the posterior wall from the neck to the dome to observe the mucosal morphology of the repair area.\u003c/p\u003e \u003cp\u003eHistological Analysis of the Compound Grafts and Retrieved Bladder\u003c/p\u003e \u003cp\u003eThe granulation tissue and the compound grafts were fixed with 10% buffered formalin and subsequently embedded in paraffin respectively. The serial sections of the tissues were stained with H\u0026amp;E and underwent immunohistochemistry analysis. The antibodies against pan-cytokeratin AE1/AE3 and vimentin were used to detect MCs, while the alpha-smooth muscle actin (α-SMA) antibody (Thermo Fisher Scientific, Waltham, USA) was used to identify myofibroblasts.\u003c/p\u003e \u003cp\u003eTo dynamically evaluate the neobladder regeneration process, the retrieved bladders at each time point were prepared for H\u0026amp;E staining and immunohistochemistry analysis, respectively, highlighting the margin between the graft and native bladder. The pan-cytokeratin AE1/AE3 antibody was used to identify the urothelium, while the α-SMA antibody was used to identify smooth muscle bundles. The immunolabeling was conducted using the avidin-biotin detection system, and the sections were counterstained with hematoxylin.\u003c/p\u003e \u003cp\u003eTransmission Electron Microscopy of the Compound Grafts\u003c/p\u003e \u003cp\u003eThe seeded grafts were trimmed to a size of 1 mm \u0026times; 1 mm \u0026times; 3 mm, fixed immediately with 3% glutaraldehyde, and then postfixed with 1% osmium tetraoxide. The specimen was then stained en bloc with 0.5% uranyl acetate and embedded in Epon 812 for sectioning. Thin sections were subsequently stained and examined using a JEOL-1200EX microscope (JEOL, Japan).\u003c/p\u003e \u003cp\u003eUrodynamics\u003c/p\u003e \u003cp\u003eUrodynamics were performed without anesthesia at 6 months postoperatively with a urodynamic system (PowerLab 4/26, Adinstruments, Australia). Each group consists of three animals. Another three animals that have not undergone bladder reconstruction surgery were selected as controls. The urethral pressure measurement tube was inserted into the bladder through urethra and the rectal pressure measurement tube was inserted into the rectum. Both tubes were then connected to the urodynamic system. Normal saline was injected into the bladder at a rate of 20 ml/min through an inflow pump. The intravesical pressure (Pves), the abdominal pressure (Pabd) and bladder capacity were monitored simultaneously. Leak point pressure(LPP) and the maximum volume of the bladder(Vol\u003csub\u003emax\u003c/sub\u003e) are defined as the Pves and the bladder capacity when the first fluid leakage was observed, respectively. Bladder compliance is defined as the ratio of Vol\u003csub\u003emax\u003c/sub\u003e to the difference between LPP and Pabd. All values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s t-test was applied for statistical analysis. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThree weeks after implantation, all tubes were fully encapsulated with translucent granulation tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Histological analysis confirmed that the tissue consisted of a wall of myofibroblasts which stained positively for α-SM actin and vimentin, and a collagen matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Transmission electron micrograph showed that the myofibroblasts were spindleshaped and contained large amounts of synthetic organelles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith the aforementioned procedure, MCs were successfully cultured in vitro and maintained their epithelial cell morphology of characteristic \"cobblestone-like\" growth pattern at the third passage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Immunochemistry staining confirmed the positive expression of both AE1/AE3 and vimentin in MCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,C).\u003c/p\u003e \u003cp\u003eThe collected MCs could be easily seeded onto and attached to the surface of the autologous granulation tissue. Histological analysis of the MCs-seeded compound graft showed a single layer of MCs forming on the outer surface of the tissue after incubation for 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E,F). The transmission electron microscopy confirmed the presence of characteristic surface microvilli and tight junctions between MCs, indicating their functional integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll animals survived the surgery. Prior to harvesting the bladder, 30 ml of normal saline was injected into the bladder for observation of the morphology and size of the repaired area. No diverticulum and urine leakage occurred in all animals. In the control group, severe adhesion was observed between the repaired area and surrounding tissues by one month after surgery. The texture of the repaired area was relatively hard (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Remarkable contracture and scarring of the grafts was observed at 2 and 6 months after implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E). Gross examination of the bladder mucosa by 1 month revealed ulcer, necotic tissue and stone formation at the repair area with no bladder mucosa regenerated on the surface of the graft (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). By 2 months, the ulcer area decreased in size and the bladder mucosa had partially covered the repair area (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Stone could still be observed. Six months after surgery, the ulcer and stone disappeared and were completely replaced by pale and stiff scar tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, in the experimental goup, no severe fibrosis and shrinkage were observed in the bladder filling state as indicated by the marking sutures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,C,E). The mucosa at the grafts site appeared sleek and normal in color with no ulcer and stone formation at different time points postoperatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,D,F). By 1 and 2 months postoperatively, the thickness of the bladder wall at the graft site was thinner than that of normal bladder. Six months postoperatively, the wall thickness of the graft increased and became similar to normal bladder.\u003c/p\u003e \u003cp\u003eThe bladder repair progress were further evaluated by histological analysis. In the control group, H\u0026amp;E staining and immunohistochemical analysis by 1 month revealed necrotic tissue on the surface of the grafts with no epithelial layers formed. Large amounts of inflammatory cells accumulated beneath the necrotic tissue. Fibroblasts were observed within the grafts especially in the margin with native bladder tissue. No smooth muscle bundles and capillaries were observed within the grafts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B,C). By 2 months, the necrotic tissue nearly disappeared, but inflammatory cells were still observed on the surface of the grafts. Urothelium began to grow into the grafts from the margin, but no complete urothelial layers had been formed. Fibroblasts subsided significantly and fibrous tissues were found within the grafts. Only a small amount of smooth muscle bundles can be seen only at the edge of the graft, although newly formed capillaries were observed within the graft (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD,E,F). By 6 months, although inflammatory cells subsided and the urothelium was well developed, only scant and disordered smooth muscle bundles were observed within the grafts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG,H,I).\u003c/p\u003e\u003cp\u003eIn the experimental group, histological analysis by 1 month showed that the grafts were fully covered by multilayered continuous epithelial layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B,C). The epithelium stained positively with antibody of AE1/AE3, but negatively with antibody of vimentin (data not shown), indicating that the original mesothelium had been completely replaced by the newly formed urothelium. Unlike the unseeded group, no significant infiltration of inflammatory cells into the grafts was observed. Sparse bundles of smooth muscle were observed to infiltrate the grafts at the site of anastomosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Neovascularization began to form in the submucosal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). By 2 months, the urothelium became more mature with more mucosal furrows. Meanwile, the density of the newly formed smooth muscle bundles and capillaries increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD,E,F). By 6 months, the way that the smooth muscle bundles were arranged became more closely aligned with the inner circular-outer longtidinal arrangement of the normal bladder (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG,H,I). The margin between the graft and native bladder became nearly indistinguishable.\u003c/p\u003e\u003cp\u003eIn addition to histological analysis, the repair effects were also evaluated by urodynamics before sampling at 6 months post-surgery. The results revealed significant differences between the normal, experimental, and control groups in terms of Vol\u003csub\u003emax\u003c/sub\u003e, LPP and bladder compliance (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The normal group had a Vol\u003csub\u003emax\u003c/sub\u003e of 86.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 ml, the experimental group 83.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08 ml, and the control group 76.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22 ml. In terms of LPP, the normal group measured 16.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55 mmHg, the experimental group 18.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 mmHg, and the control group 24.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22 mmHg. Bladder compliance was highest in the normal group at 6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 ml/mmHg, followed by the experimental group at 5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 ml/mmHg, and the control group at 3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 ml/mmHg.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the last two decades, tissue engineering has been focusing on bladder tissue reconstruction. Although much progress has been achieved in this field, most studies still remains confined to the stage of experimental research. One of the key elements for successful repair is the appropriate selection of seed cells. From the perspective of the histological structure of the urinary system, urothelial cells and smooth muscle cells are ideal sources of seed cells, and some successful experiences have been gained in animal experiment studies[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, there are some limitations such as difficulties in in-vitro culture and slow expansion speed of urothelial cells, which makes it difficult to ensure the synchronous expansion of the two types of seed cells in vitro, leading to a longer period and higher failure rate of tissue construction. Moreover, when a patient is accompanied by urothelial tumor, autologous urothelial cells are not suitable for use as seed cells. These limitations have become important factors that impede the clinical applications of tissue engineering for reconstruction of the urinary tract. Therefore, it is necessary to find a substitute source of seed cells that is plentiful, easy to obtain, easy to survive and expand in vitro, and suitable for urinary tract repair.\u003c/p\u003e \u003cp\u003eDespite originating from the mesoderm, MCs display epithelial cell growth patterns and express epithelial cell markers[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In patients with peritoneal dialysis, despite continuous exposure to dialysate, there was no significant damage or fibrosis of the peritoneum, indicating the good tolerance of MCs to similar components to urine. Peritoneum has also been attempted as a graft for urethra repair in experimental research, and the results were promising [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our previous studies[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], we applied MCs as seed cells to repair urethra defect and the seeded MCs successfully prevented graft fibrosis and contracture, demonstrating that MCs could serve as impermeable barriers to harmful urine components. In the present study, we further investigated the feasibility of using MCs for bladder reconstruction. Although the bladder mucosa has a longer contact time with urine as a storage organ, promising results were found. In the control group, we observed a tissue repair process similar to that after transurethral resection of bladder tumors (TURBt), where necrotic tissue or stones first formed on the surface, followed by formation of scar tissue. By contrast, no signs of excessive accumulation of inflammatory cells and fibrosis were observed in the grafts seeded with MCs. We observed that the inflammatory cells accumulated mainly on the surface of the graft in the unseeded group, indicating that urine infiltration was the major cause. Our findings further confirmed that the seeded MCs could form an effective mucosal barrier for bladder repair. Moreover, MCs also possess anti-inflammatory properties. Studies have shown that MCs can effectively inhibit the proliferation of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T lymphocytes by secreting anti-inflammatory factor TGF-β[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another in vitro study showed that MCs consume L-arginine through the secretion of arginase I, which subsequently inhibits the activation of CD4\u0026thinsp;+\u0026thinsp;lymphocytes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As seed cells, the anti-inflammatory properties of MCs might contribute to reducing the inflammatory response at the repair site, which was beneficial for tissue regeneration and repair.\u003c/p\u003e \u003cp\u003eInterestingly, we observed that urothelium in the seeded grafts regenerated much faster than that in the unseeded grafts. It is still unclear how the MCs promoted the regeneration of uroepithelial cells. It has been proved that MCs can be transformed into smooth muscle cells, myofibroblasts, endothelial cells, and even bone and cartilage tissue under specific in-vivo environments, participating in the development or reconstruction of tissue and organs [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In-vitro studies have also confirmed that MCs can be successfully induced to differentiate into osteoblasts, adipocytes, and smooth muscle cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In view of the pluripotent nature of MCs, there is the possibility that MCs underwent transdifferentiation into urothelial cells under the stimulation of urine-based environment. However, it might also be possible that MCs just sloughed off the grafts and were gradually replaced by local urothelium, considering the capability of the urothelium to migrate and proliferate[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The basement membrane or growth factors secreted by MCs may accelerate the migration of surrounding urothelial cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The formation of complete urothelial layers over the grafts, in turn, promoted the regeneration of smooth muscle bundles, as the urothelium is thought to play a major role in the differentiation and maintenance of the bladder muscle layer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Anyway, the destiny of MCs after implantation and how MCs promoted urothelium regeneration needs to be further investigated.\u003c/p\u003e \u003cp\u003eIn addition to seed cells, the selection of scaffold materials is also critical to the success of tissue repair. An ideal scaffold material should possess good cell compatibility, mechanical strength, and degradability[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Acellular matrix is commonly used as a scaffold material for urinary tract tissue engineering, including bladder acellular matrix, small intestinal submucosa layer acellular matrix, vascular acellular matrix, and et al[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the preparation process is time-consuming, and continuous agitation and enzyme digestion can damage the mechanical properties of the scaffold. Additionally, incomplete acellularization can lead to immune reactivity and induce local inflammatory reactions, which is harmful to tissue repair. In the present study, we implanted silicone tubes into the subcutaneous tissue of animals and induced the formation of an autologous granulation tissue, mainly composed of myofibroblasts and collagen matrix. This tissue is of fully autologous origin, without any immunogenicity. It does not require decellularization processing, and thus its mechanical properties and biological activity can be well preserved. Another feature of this tissue is that it resembles corneal tissue with no vascular growth inside, and that its cells' activity fully relies on the nutrients provided by the surrounding tissue fluid. This suggests that it may have better tolerance to the ischemic environment during the initial period of urinary tract substitution. This has been confirmed in our previous study[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], in which we successfully applied this kind of tissue in the repair of long urethral defect.\u003c/p\u003e \u003cp\u003eFor the tissue engineering construction of muscular organs, in addition to seeding epithelial cells, it is necessary to concurrently or sequentially seed cells that produce extracellular matrix, such as smooth muscle cells, fibroblasts, and mesenchymal stem cells, to improve the mechanical properties and biocompatibility of the graft[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This autologous granulation tissue is already rich in myofibroblasts, so only epithelial cells need to be seeded, which shortens the preparation period, simplifies the construction process and reduces the cost. Myofibroblasts have a morphology and function similar to smooth muscle cells. In the present study, myofibroblasts were observed to be completely replaced by smooth muscle cells after being used for bladder repair. Previous studies have suggested that myofibroblasts can be converted into smooth muscle cells under stimulation of cyclic mechanical stretch[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], while others have proposed that myofibroblasts secrete chemotactic factors that induce host smooth muscle cells to migrate into the graft[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. One month after surgery, we observed the coexistence of myofibrioblasts and smooth muscle bundles within the seeded grafts. Two months after surgery, myofibroblasts were completely replaced by smooth muscle cells. However, we also observed that newly formed smooth muscle bundles firstly appeared at the edges of the graft and then migrated towards the center over time. Therefore, we speculated that both mechanisms were involved in smooth muscle regeneration. Regardless of the mechanism, our study confirmed that the granulation tissue sucessfully promoted smooth muscle regeneration in the presence of an effective epithelial barrier, which was crucial to maintain the structural and functional integrity of the bladder. This was also reflected by the results of urodynamics, which showed the experimental group had a larger bladder capacity and better bladder compliance. The exact mechanism by which myofibroblasts promote smooth muscle regeneration needs to be fully elucidated in the future study.\u003c/p\u003e \u003cp\u003eThere were several limitations in this study. First, the bladder defect was created in normal healthy bladder, which could not fully simulate the clinical situation. Second, the number of experimental animals is relatively small. To precisely evaluate the success rate, larger sample sizes are needed. Third, as a small-bodied animal, rabbit may not provide as much information for clinical problems. Large-scale animal research is necessary in the future. Finally, as a preliminary study, we have not addressed the repair of a significant bladder defect. The main aim of the study was to evaluate the ability of the constructed tissue to regenerate bladder tissue. In the future study, we will investigate the effect of the constructed tissue for larger bladder defect repair.\u003c/p\u003e \u003cp\u003eIn summary, we successfully constructed a kind of compound grafts with an inner mesothelium, and a contractile wall of myofibroblasts and collagens. They could guide bladder regeneration for a defect of 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e in male rabbits. Considering clinical applications, the human scrotum might be a suitable option for mold implantation, which resembles subcutaneous tissue of rabbits with a lack of fat tissue. It might become a promising technique in the clinical use for bladder reconstruction in human after further investigations.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur research has demonstrated that granulation tissue seeded with mesothelial cells can serve as an effective material for bladder patch repair in male rabbits. This approach leverages the unique properties of mesothelial cells, which include their ability to promote tissue regeneration and healing. In studies conducted on male rabbits, the mesothelial cell-seeded granulation tissue was observed to integrate well with the native bladder tissue, facilitating the restoration of bladder functionality and maintaining structural integrity. These findings suggest the potential for this technique to provide a biocompatible and efficient alternative for bladder repair, opening avenues for further exploration in regenerative medicine and potential applications in clinical settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the grants from Natural Science Foundation of Shandong Province (ZR2021MH049)\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eL.Y. and Z.L. wrote the manuscript. G.G. planned the study. L.Y., Y.S., L.Q. and Y.S contributed to analysis and discussion of the data. J.Z., J.S., and Q.C. analyzed the results. G.G supervised the project. All authors participated in animal experiments.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNatural Science Foundation of Shandong Province.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eNo datasets were generated or analysed during the\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHorst M, Eberli D, Gobet R, Salemi S (2019) Tissue Engineering in Pediatric Bladder Reconstruction-The Road to Success. Front Pediatr 7:91\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoth JD, Cain MP (2018) Neuropathic Bladder and Augmentation Cystoplasty. Urol Clin North Am 45:571\u0026ndash;585\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDougal WS (1992) Metabolic complications of urinary intestinal diversion. 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Cell Death Dis 5:e1304\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerrick SE, Mutsaers SE (2004) Mesothelial progenitor cells and their potential in tissue engineering. Int J Biochem Cell Biol 36:621\u0026ndash;642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaskin LS, Hayward SW, DiSandro MS, Li YW, Cunha GR (1999) Epithelial-mesenchymal interactions in the bladder. Implications for bladder augmentation. Adv Exp Med Biol 462:49\u0026ndash;61\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasarin M, Morlacco A, Dal Moro F (2021) Bladder Substitution: The Role of Tissue Engineering and Biomaterials. Processes; 9:1643\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang SW, Xu ZH, Zhao YY, Yan L, Zhou ZL, Gu GL (2018) Autologous granulation tissue tubes for replacement of urethral defects: An experimental study in male rabbits. J Pediatr Urol; 14:14.e11-14.e17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoys AJ, Barron SL, Tilev D, Owens RM (2020) Building Scaffolds for Tubular Tissue Engineering. Front Bioeng Biotechnol 8:589960\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouhout S, Chabaud S, Bolduc S (2016) Organ-specific matrix self-assembled by mesenchymal cells improves the normal urothelial differentiation in vitro. World J Urol 34:121\u0026ndash;130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrewa T (2007) The artificial conduit for urinary diversion in rats: a preliminary study. Transpl Proc 39:1647\u0026ndash;1651\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell GR, Turnbull G, Xiang L et al (2008) The peritoneal cavity as a bioreactor for tissue engineering visceral organs: bladder, uterus and vas deferens. J Tissue Eng Regen Med 2:50\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhargava S, Patterson JM, Inman RD, MacNeil S, Chapple CR (2008) Tissue-engineered buccal mucosa urethroplasty-clinical outcomes. Eur Urol 53:1263\u0026ndash;1269\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjur","sideBox":"Learn more about [World Journal of Urology](https://link.springer.com/journal/345)","snPcode":"345","submissionUrl":"https://submission.nature.com/new-submission/345/3","title":"World Journal of Urology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5736210/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5736210/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eCurrently, bladder reconstruction using gastrointestinal segments is considered as the gold standard for bladder repair or augmentation. However, postoperative complications including bowel dysfunction, metabolic abnormalities, chronic urinary tract infection and stone formation are common. This study aims to evaluate the utility of the compound graft for bladder patch repair by seeding mesothelial cells onto autogenous granulation tissue.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e22 Fr silastic tubes were implanted subcutaneously in 18 male rabbits. The animals were equally divided into two groups. In the experimental group, nine animals underwent omentum biopsies and mesothelial cells were cultured and expanded in vitro. Two weeks after the implantation, the autologous granulation tissue encapsulating the silastic tubes were harvested. The tissue was cut longitudinally and trimmed to 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e. In the experimental group, mesothelial cells were seeded onto the outer surface of the tissue and cocultured for 7 days to construct a compound graft. In each animal, a full-thickness defect of 2\u0026times;2 cm\u003csup\u003e2\u003c/sup\u003e in the anterior wall of the bladder was created. The defect was repaired with the constructed compound graft in the experimental group and with the granulation tissue directly in the control group. The bladder was harvested and analyzed grossly and histologically at 1, 2 and 6 months postoperatively. In addition, urodynamics were performed 6 months postoperatively to evaluate the function of the bladder.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the experimental group, no severe fibrosis and shrinkage were observed postoperatively and the mucosa at the grafts site appeared sleek and normal in color. Histologically, multilayers of urothelium surrounded by increasingly organized smooth muscles were observed. In contrast, severe contracture and fibrosis of the grafts occured in the control group. Gross examination of bladder mucosa revealed ulcer and stone formation at the site of the grafts. Histologically, accumulation of inflammatory cells and fibroblasts, and extensive scarring occurred. Six months after surgery, the urodynamic results showed that the experimental group had a larger bladder capacity, a lower maximum intravesical pressure and a better bladder compliance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMesothelial cell-seeded granulation tissue can be successfully used for bladder patch repair in male rabbits.\u003c/p\u003e","manuscriptTitle":"Bladder Patch Repair Using Mesothelial Cell-Seeded Autologous Granulation Tissue: An Experimental Study in Male Rabbits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-02 18:22:21","doi":"10.21203/rs.3.rs-5736210/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-04T03:21:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-27T21:39:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333756508398854396570855864187406607839","date":"2025-01-19T17:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336038260817427474491442045940485884541","date":"2025-01-18T12:47:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163478302283819722543962596732533194041","date":"2025-01-15T04:27:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-08T17:36:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100567577922441497166937283391169342903","date":"2024-12-31T19:03:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-31T15:56:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-31T14:53:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-31T11:50:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Urology","date":"2024-12-30T14:18:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjur","sideBox":"Learn more about [World Journal of Urology](https://link.springer.com/journal/345)","snPcode":"345","submissionUrl":"https://submission.nature.com/new-submission/345/3","title":"World Journal of Urology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"733bdfc6-67f4-4016-89fe-167da7003d51","owner":[],"postedDate":"January 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T15:57:56+00:00","versionOfRecord":{"articleIdentity":"rs-5736210","link":"https://doi.org/10.1007/s00345-025-05611-7","journal":{"identity":"world-journal-of-urology","isVorOnly":false,"title":"World Journal of Urology"},"publishedOn":"2025-05-07 15:56:50","publishedOnDateReadable":"May 7th, 2025"},"versionCreatedAt":"2025-01-02 18:22:21","video":"","vorDoi":"10.1007/s00345-025-05611-7","vorDoiUrl":"https://doi.org/10.1007/s00345-025-05611-7","workflowStages":[]},"version":"v1","identity":"rs-5736210","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5736210","identity":"rs-5736210","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}