Recent trends in 3D bioprinting technology for skeletal muscle regeneration.

Skeletal muscle is a pro-regenerative tissue, that utilizes a tissue-resident stem cell system to effect repair upon injury. Despite the demonstrated efficiency of this system in restoring muscle mass after many acute injuries, in conditions of severe trauma such as those evident in volumetric muscle loss (VML) (>20 % by mass), this self-repair capability is unable to restore tissue architecture, requiring interventions which currently are largely surgical. As a possible alternative, the generation of artificial muscle using tissue engineering approaches may also be of importance in the treatment of VML and muscle diseases such as dystrophies. Three-dimensional (3D) bioprinting has been identified as a promising technique for regeneration of the complex architecture of skeletal muscle. This review discusses existing treatment strategies following muscle damage, recent progress in bioprinting techniques, the bioinks used for muscle regeneration, the immunogenicity of scaffold materials, and in vitro and in vivo maturation techniques for 3D bio-printed muscle constructs. The pros and cons of these bioink formulations are also highlighted. Finally, we present the current limitations and challenges in the field and critical factors to consider for bioprinting approaches to become more translationa and to produce clinically relevant engineered muscle. STATEMENT OF SIGNIFICANCE: This review discusses the physiopathology of muscle injuries and existing clinical treatment strategies for muscle damage, the types of bioprinting techniques that have been applied to bioprinting of muscle, and the bioinks commonly used for muscle regeneration. The pros and cons of these bioinks are highlighted. We present a discussion of existing gaps in the literature and critical factors to consider for the translation of bioprinting approaches and to produce clinically relevant engineered muscle. Finally, we provide insights into what we believe will be the next steps required before the realization of the application of tissue-engineered muscle in humans. We believe this manuscript is an insightful, timely, and instructive review that will guide future muscle bioprinting research from a fundamental construct creation approach, down a translational pathway to achieve the desired impact in the clinic.

Ex-Vivo and In-Vivo Expansion of Spermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model.

AIMS: This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs). METHODS: For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation. RESULTS: Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds. CONCLUSION: Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.

Meatal stenosis following three types of circumcision with frenular artery preservation (FAP), the Plastibell device (PD), and frenular artery ligation (FAL): a long-term follow-up.

BACKGROUND: Despite the simplicity of male circumcision, complications occur frequently. Post-circumcision meatal stenosis is a concerning complication that might require several interventions. AIM: This study aims to evaluate the incidence of meatal stenosis in long-term follow-up, following three common circumcision methods: frenular artery preservation, frenular ligation, and the Plastibell device. METHODS: This study is the continuation of the previous randomized clinical trial, the preliminary abstract of which has been accepted in the annual meeting of the American Urological Association in 2011. However, in this paper, we only included the patients with results of long-term follow-up. Patients were followed for a median of 11 years (range, 7-17). Follow-ups were recorded by evaluation of meatus and signs and symptoms of meatal stenosis. RESULTS: Two hundred six boys (80 neonates and 126 non-neonates) at the time of procedure were included in this study. The circumcision was conducted on 23.3% (48/206) of boys with the Plastibell device (PD) and 39.3% (81/206) of cases with frenular artery preservation (FAP) and 37.4% (77/206) of cases with frenular artery ligation (FAL). Meatal stenosis presented in 13 children during follow-up. Considering the three methods of circumcision, a significant difference in the incidence of meatal stenosis among the types of circumcisions was observed (6.3% in PD and 1.2% in FAP, 11.7% in FAL, P = 0.026). CONCLUSION: The present study revealed that the technique preserving the frenular artery is associated with a significantly lower incidence of meatal stenosis. Hence, the FAP is the recommended technique for circumcision as compared to two other methods.

Insights and outcomes of single-staged repair of female bladder exstrophy-epispadias complex without osteotomy: 15 Years experience of a single institution.

INTRODUCTION: Female exstrophy-epispadias complex (BEEC) has been considered as a rare malformation of the genito-urinary tract affecting. Combining procedures during the reconstruction of bladder exstrophy-epispadias complex to reduce the number of procedures and improve the outcomes has evoked great interest. OBJECTIVE: we tried to describe the application and results of a single-stage approach for reconstruction of female BEEC during initial reconstruction or following prior failed bladder closure (FBC). STUDY DESIGN: The records of 37 female patients referred for the repair of BEEC without the application of pelvic osteotomies were extracted from an institutionally approved database from September 2002 to August 2018. The mean patient age was 7.24 and 26 patients had a prior FBC. All patients underwent pelvic floor electrical stimulation and toilet training for 1 year after the closure. Complete continence was defined as having the ability to stay dry for more than 3 h without leakage during the day and night. Partial continence has traditionally been defined as retaining urine for 1-3 h or having some stress incontinence. Incontinence was defined as a continence interval of less than 1 h. RESULTS: None of the patients presented bladder prolapse or dehiscence on follow-up; while stricture developed in 2 patients (5.4%). A total of 25 (67.6%) children were dry during the day and night. However, 9 (24.3%) were dry during the day but wet at night; while 3 (8.1%) were totally incontinent. The patients were followed up for a mean of 112.56 months. DISCUSSION: Although earlier reports of this technique seem encouraging, it should be mentioned that postoperative complications are possible and difficult to manage. However, none of our patients were presented with severe postoperative complications in the follow-ups. CONCLUSION: The single-stage technique provides satisfactory outcomes in selected patients with classic bladder exstrophy. The majority of patients attained social dryness without bladder augmentation and intermittent catheterization accompanied with minimum complication rate and best cosmetic results.

Regeneration of muscular wall of the bladder using a ureter matrix graft as a scaffold.

We investigated a method for bladder augmentation in rats using a decellularized ureter graft. We used 16 rats divided into two groups of eight. After partial cystectomy, the bladders in group 1 were grafted with a 1 cm2 patch of human decellularized ureter. Rats in group 2 were untreated controls. Biopsies of the graft were taken at 1, 3 and 9 months postoperatively for histological investigation. Total removal of cells and preservation of extracellular matrix (ECM) was confirmed in the decellularized ureter. Histological examination after 1 month revealed few cells at the border of the graft. Three months after the operation, the graft was infiltrated by vessels and smooth muscle and the mucosal lining was complete. All bladder wall components resembled native bladder wall by 9 months after implantation. CD34, CD31, α-smooth muscle actin, S100, cytokeratin AE1/AE3 and vimentin were detected 9 months after the operation. We demonstrated the potential of decellularized biocompatible ureteric grafts for use as a natural collagen scaffold for bladder repair in rats.

Revascularized Pyelo-Uretero-Cystoplasty in Patients with Chronic Bladder Outlet Obstruction Due to Ectopic Ureterocele: A Safe Surgical Technique with Superior Continence Outcomes.

PURPOSE: To present the outcomes of revascularized pyeloureterocystoplasty with ureterocele unroofing in end stage bladder patients with duplex system and ureterocele. METHODS: Thirteen patients with obstruction of intrauterine outlet from an ectopic obstructive ureterocele were included. Fourteen units of duplex systems underwent upper pole partial nephrectomy in conjunction with augmentation revascularized pyeloureterocystoplaty and ureterocele unroofing. The anterior and lateral walls of the ureterocele were excised before cystoplasty, and the resultant edges of the posterior wall were sutured to the bladder epithelium. Anastomosis of the upper pole vein and artery to the inferior iliac artery and the common iliac vein was performed. Detubularization of the whole ureter was performed with exception of the intramural ureteric part that kept tubularized for 'jet/turbulent' occurrence. Five patients (control group) underwent pyeloureterocystoplasty without revascularization. Patients underwent several evaluations in long-term follow-up. RESULTS: Patients were all dry by day and night in our long-term follow-up. Urinary incontinence improved in patients with no need for re-augmentation technique. Vesicoureteral reflux subsided in all patients postoperatively except one, who was asymptomatic. After five years, median bladder capacity rose from 128.5 ml to 395 ml and bladder compliance showed significant improvement from 15 ml/cm H2O to 29 ml/cm H2O, in experimental group and remained stable for 24-36 months. Median bladder capacity did not rise significantly in control group. CONCLUSION: Pyeloureterocystoplasty is an efficient choice in this type of patients, which may prevent the recurrence of hypocompliant bladders and prevent ischemia and subsequent fibrosis.

Lung Extracellular Matrix as a Platform for Lung Organ Bioengineering: Design and Development of Tissue Engineered Lung.

Since lung tissue is not able to be reconstructed after substantial injury, lung transplantation often is the only alternative for treatment. Antibiotic-resistant organisms that remain in donor lungs causing infection in the immunosuppressed recipient are among the complications following transplantation. Development of strategies for whole lung regeneration is a pleasing choice particularly in patients with end-stage lung diseases. Reconstruction of lung tissue in vitro for transplantation received increased attention which could deal with the shortage of donor organs. Recent advancements in the field of tissue engineering and regenerative medicine have paved the road for beneficial alternative therapies. Our group has extensive experience with regard to the structure of the lung tissue, which makes us to our decision to continue with the preparation of lung, with the aim of developing a new ECM scaffold. Herein, we aim to review the state-of-art and the tissue engineering and regenerative medicine technology highlighting the major achievements toward the production of a bioengineered lung obtained decellularization and recellularization techniques. We have strong hopes that recent developments in the engineering of lung will lead to similar breakthroughs in the engineering of distal lung components in the future.

Cardiac Extracellular Matrix as a Platform for Heart Organ Bioengineering: Design and Development of Tissue-Engineered Heart.

The field of tissue engineering and regenerative medicine is able to depict the mechanism of cardiac repair and development of cardiac function as well, in order to reveal findings to new therapeutic designs for clinical treatment. The foremost approach of this scientific field is the fabrication of scaffolds, which contain cells that can be used as cardiac grafts in the body, to have the preferred recovery. Cardiac tissue engineering has not been completely organized for routine clinical usages. Hence, engineering innovations hold promise to character research and treatment options in the years to come. Our group has extensive experience with regard to the structure of the heart, which makes us to our decision to continue with the preparation of heart, with the aim of developing a new ECM scaffold. Herein, we aim to assess the state-of-the-art fabrication methods, advances in decellularization and recellularization techniques. We also highlight the major achievements toward the production of a bioengineered heart obtained from decellularization and recellularization techniques.

The Most Ideal Pancreas Extracellular Matrix as a Platform for Pancreas Bioengineering: Decellularization/Recellularization Protocols.

Natural scaffold appears to have extensive functions in providing anchorage and structural requirements, as well as providing a structural support for cell adherence and cell interaction for further recellularization process. Specific methods used for decellularization process play an essential role in the efficacy of cell removal and successful preservation of ultrastructure and biomechanical properties of the tissue. Numerous scaffolding materials and fabrication techniques have been investigated for pancreatic tissue engineering. Techniques of casting, freeze drying, injection molding, and electrospinning have been also used to fabricate scaffolds. Herein, we aim to review the state-of-the-art and the tissue engineering and regenerative medicine technology highlighting the major achievements toward the production of a bioengineered pancreas obtained decellularization techniques and cell-on-scaffold technology.

The Renal Extracellular Matrix as a Supportive Scaffold for Kidney Tissue Engineering: Progress and Future Considerations.

During the past decades, diverse methods have been used toward renal tissue engineering in order to replace renal function. The goals of all these techniques included the recapitulation of renal filtration, re-absorptive, and secretary functions, and replacement of endocrine/metabolic activities. It is also imperative to develop a reliable, up scalable, and timely manufacturing process. Decellularization of the kidney with intact ECM is crucial for in-vivo compatibility and targeted clinical application. Contemporarily there is an increasing interest and research in the field of regenerative medicine including stem cell therapy and tissue bioengineering in search for new and reproducible sources of kidneys. In this chapter, we sought to determine the most effective method of renal decellularization and recellularization with emphasis on biologic composition and support of stem cell growth. Current barriers and limitations of bioengineered strategies will be also discussed, and strategies to overcome these are suggested.

Extracellular Matrix Scaffold Using Decellularized Cartilage for Hyaline Cartilage Regeneration.

The repair of osteochondral defects is among the top ten medical needs of humans in the 21st centuries with many countries facing rapidly aging population involved with osteoarthritis as a major contributor to global disease burden. Tissue engineering methods have offered new windows of hope to treat such disorders and disabilities. Regenerative approaches to cartilage injuries require careful replication of the complex microenvironment of the native tissue. The decellularized hyaline cartilage derived from human allografts or xenografts is potentially an ideal scaffold, simulating the mechanical and biochemical properties, as well as biological microarchitecture of the hyaline cartilage. There have been many attempts to regenerate clinically viable hyaline cartilage tissue using decellularized cartilage-derived extracellular matrix with stem cell technology. This chapter describes the reproducible methods for hyaline cartilage decellularization and recellularization. In addition, quality control and characterization requirements of the product at each step, as well as the clinical applications of final product have been discussed.

Local tissue reaction and histopathological characteristics of three different bulking agents: a rabbit model

ABSTRACT Purpose: We assessed the efficacy and safety of a single injection of three bulking agents over the short- and long-term follow-ups in rabbits. Dermal and preputial matrices were compared with Deflux (DxHA) injection. Material and methods: Twenty-four rabbits were divided into three groups. Group I (n=8) underwent the injection of a lyophilized dermal matrix (LDM) beneath the seromuscular layer of the bladder wall. Rabbits in group II (n=8) were injected with lyophilized preputial matrix (LPM). Rabbits of group III (n=8) were injected with DxHA as the control group. They were followed up for 1 and 6 months after the injection. Subcutaneous injection of all bulking agents was also performed in nude mice. Biopsies were stained with LCA (leukocyte common antibody), CD68, CD31, and CD34. Scanning electron microscopy (SEM) and MTT assay were also performed. Results: Immunohistochemistry staining with CD68 and LCA revealed higher inflammation grade in LDM as compared with LPM and DxHA. Fibrosis grade was also higher in LDM both in short- and long-term follow-ups. However, no significant difference was detected in CD31 and CD34 staining between control and experimental groups. SEM analysis showed that the particle size of LPM was more similar to DxHA. MTT assay revealed that cell proliferation was similar in DxHA, LDM, and LPM. In-vivo assay in nude mice model showed more promising results in LPM as compared with LDM. Conclusion: The long-term results demonstrated that LPM was more similar to Deflux with the least local tissue reaction, inflammation, and fibrosis grade.

Intravesical electromotive administration of botulinum toxin type A in improving the bladder and bowel functions: Evidence for novel mechanism of action.

Objective: To examine the hypothesis that what is the concomitant mechanism of action botulinum toxin type A (BoNTA) administration by intravesical electromotive into the bladder resulting in bladder function improvement. We also tried to confirm the possibility of retrograde trans-axonal transportation of toxin.Design: Animal study.Setting: Ten male rabbits were divided into two groups.Participants: Group 1 (G1) (n = 5) (BoNTA/EMDA), and group 2 (G2) (n = 5) the control group.Interventions: Animals in G1received 10 IU/Kg of intravesical BoNTA through a specific catheter for electromotive drug administration (BoNTA/EMDA). About 0.1-0.15 ml of toxin was diluted in 1 ml of distilled water. The maximum frequency of the device for drug solution delivery was set at 4 mA for 15 min. In G2 as the control group, the same procedure was performed to deliver normal saline to the bladder.Outcome measures: Multiple biopsies were taken from bladder's contiguous structures one month postoperatively. The immunohistochemical (IHC) evaluation was performed with anti-clostridium botulinum toxoid type A mouse IgM monoclonal antibody.Results: In specimens of G1, BoNTA penetrated through muscular layers of the bladder wall and the staining was uniform in the urothelium, interstitium, and muscular layers. Positive IHC staining showed that BoNTA was traced in the upper and lower spinal cord in addition to pelvic nerve, sacral nerve plexus, intestine wall, and pelvic floor muscle. In G2, all the specimens were intact in IHC staining.Conclusions: The presence of BoNTA in lower and upper spinal cord suggests the possibility of retrograde trans-axonal transfer of toxin to lower and upper neural pathways which may result in simultaneous improvement in bladder and bowel functions.

Single-staged male bladder exstrophy-epispadias complex reconstruction with pubic bone adaptation without osteotomy: 15-year single-center experience.

PURPOSE: To represent the 15 years' experience of an academic referral center for the reconstruction of bladder exstrophy-epispadias complex with a modified single-stage approach. Single-staged reconstruction techniques are commonly used for classic bladder exstrophy. However, combined bladder closure and epispadias repair have been taken into great consideration in patients with initially failed reconstruction or delayed primary closure. METHODS: A total of 49 boys underwent 1-stage bladder and epispadias repair with pubic bone adaptation and without the application of pelvic osteotomy. The mean ± SD age at surgery was 5.23 ± 2.04 months. Continence and social dryness were assessed in the follow-ups with 3 months intervals for the first year and biannually thereafter. RESULTS: The mean ± SD of follow-up was 127.25 ± 71.32 months. Urethrocutaneous fistula, stricture, wound infection, and hemiglans were developed in six distinct patients. However, no other major complications were noted. Three patients (6.1%) remained incontinent; while 32 (65.3%) children were socially continent and 14 (28.6%) children were waiting for toilet training. All the patients without previous failed closure were socially continent, while all incontinent patients had two failed closures in their history. No patient was rendered hypospadiac. CONCLUSION: Based on the experience of this institution, the application of single-stage reconstructive techniques can lead to continence, cosmetically pleasing appearance with promising outcomes, and reduction of overall operations, hospital stay and costs in the majority of cases as compared to multiple surgical procedures.

Management of urinary and bowel dysfunction in rabbit model of spinal cord injury using Schwann cells and muscle progenitors: functional study and evidence for novel mechanism of action.

PURPOSE: We tried to investigate the role of Schwann and satellite cells in the treatment of neurogenic bladder and bowel dysfunction; following spinal cord injury in the rabbit model. METHODS: Twelve male New Zealand rabbits underwent induction of neurogenic bladder by spinal cord injury. Rabbits underwent the fiber tractography analysis to confirm the induction of spinal cord injury. Then, animals were randomly divided into two groups. In group I (n = 4), Schwann cells were obtained from autologous peroneal nerve. In group II (n = 4), the co-culture of nerve-muscle cells was obtained from autologous peroneal nerve and quadriceps muscle. Animals in the control group (n = 4) did not undergo any rehabilitation therapy. One and 4 months after injection of cells into the external anal sphincter, electromyography, urethral pressure profiles, urodynamic studies, voiding cystourethrogram, and manometry was performed to confirm the efficacy of treatment in short- (1 month) and long-term (4 months) follow-ups. RESULTS: The investigations validated that no statistically significant difference was detected between the two experimental groups in a short-term follow-up (p-value > 0.05). However, the functional features were improved in group II in long-term follow-up. In both groups, the external anal sphincter contracted in response to electrical signals delivered to the muscle. However, more signals were detected in group II in electromyography evaluation. The immunohistochemical staining demonstrated that the histological features of the bladder and spinal cord were more satisfactory in group II in all follow-ups compared to group I, in terms of less edema, inflammation, presence of progenitor cells, and expression of muscle and nerve markes. CONCLUSION: Our results suggested that the injection of nerve-muscle co-culture cells into the external anal sphincter may be a helpful tactic for ameliorating the urological complications; following spinal cord injury induction in the rabbit model.

Recellularization of testicular feminization testis in C57bl6 as a natural bioreactor for creation of cellularized seminiferous tubules: an experimental study.

We determined histological aspects of implanted human decellularized testicular matrix (DTM) in C57BL6 as a primitive step for further testis tissue engineering. A total of 4 immature human testicles were obtained after bilateral orchiectomy from patients with testicular feminization syndrome. The optimal decellularization protocol was determined and the efficacy of decellularization was evaluated in two of the testicles. The remaining scaffolds were cut in 3 × 3 mm3 pieces and implanted between the tight muscles in 32 C57BL6. Biopsies were taken at 2, 4, 8, and 24 weeks postoperatively and stained with PLZF, protamine, and tekt1 markers. Histological examination of DTMs confirmed complete absence of nuclear remnants and preservation of the extracellular matrix. Successful cell seeding was observed in all follow-ups confirmed by H&E and IHC staining that increased continuously during the whole study. Interestingly, spermatogonial stem-like cells were observed on decellularized implants that were well differentiated during the follow-ups. Natural bioreactors may provide a good cell source for testes tissue regeneration. This technique may provide testis bioscaffold as a three-dimensional platform and further successful cell seeding to produce a functional testis. This novel technique may be beneficial for patients who require testicular supplementation.

Local tissue reaction and histopathological characteristics of three different bulking agents: a rabbit model.

PURPOSE: We assessed the efficacy and safety of a single injection of three bulking agents over the short- and long-term follow-ups in rabbits. Dermal and preputial matrices were compared with Deflux (DxHA) injection. MATERIAL AND METHODS: Twenty-four rabbits were divided into three groups. Group I (n=8) underwent the injection of a lyophilized dermal matrix (LDM) beneath the seromuscular layer of the bladder wall. Rabbits in group II (n=8) were injected with lyophilized preputial matrix (LPM). Rabbits of group III (n=8) were injected with DxHA as the control group. They were followed up for 1 and 6 months after the injection. Subcutaneous injection of all bulking agents was also performed in nude mice. Biopsies were stained with LCA (leukocyte common antibody), CD68, CD31, and CD34. Scanning electron microscopy (SEM) and MTT assay were also performed. RESULTS: Immunohistochemistry staining with CD68 and LCA revealed higher inflammation grade in LDM as compared with LPM and DxHA. Fibrosis grade was also higher in LDM both in short- and long-term follow-ups. However, no significant difference was detected in CD31 and CD34 staining between control and experimental groups. SEM analysis showed that the particle size of LPM was more similar to DxHA. MTT assay revealed that cell proliferation was similar in DxHA, LDM, and LPM. In-vivo assay in nude mice model showed more promising results in LPM as compared with LDM. CONCLUSION: The long-term results demonstrated that LPM was more similar to Deflux with the least local tissue reaction, inflammation, and fibrosis grade.

Bladder Herniation as an Auto-Augmentation Technique in Bladder Exstrophy: Initial Experience in Patients with Small Bladder Plate.

OBJECTIVE: To present our long-term experience of bladder plate herniation technique in patients with bladder exstrophy epispadias complex (BEEC) and inadequate bladder plate. METHODS: Ten BEEC patients with inadequate bladder plates were referred. The bladder underlying fascia was opened and the exstrophic bladder was fixed above the peritoneal cavity to herniate the peritoneal contents beneath the bladder plate so that the abdominal pressure would be directly transferred to the posterior bladder wall; causing gradual bladder expansion and auto-augmentation. In 5 patients, the inguinal hernia was fixed to increase the pressure transferred to the exstrophic bladder. The bladder capacity was measured while the patient was crying and when the bladder was enlarged. Cystometry and voiding cystourethrogram were performed before the application of this technique and during the next 6 to 8 months, to determine the bladder capacity for further primary bladder closure. RESULTS: The bladder was enlarged during straining/crying without any complications. The average bladder capacity was increased about 2.5 to 3 times after 8 months of follow-up so that it was suitable for undergoing primary closure. None of the children needed bladder augmentation following the single-stage total BEEC reconstruction. CONCLUSION: This technique seems to be safe, effective, and feasible in patients with small-sized bladder and may be performed before the primary closure to increase the success rate. This technique may be effective in increasing the bladder capacity for staged bladder closure and bladder neck reconstruction without further need for bladder augmentation.

In-vivo regeneration of bladder muscular wall with whole decellularized bladder matrix: A novel hourglass technique for duplication of bladder volume in rabbit model.

OBJECTIVE: To determine histological aspects of decellularized bladder graft to achieve a double-sized bladder by novel hourglass technique; using rabbit models. METHODS: Sixteen rabbit bladders were decellularized and underwent laboratory investigations. After making a laparotomy incision and exposure of bladders in another 16 rabbits (partial detrusor myomectomy), they were separated into two groups. The fundus of the decellularized scaffold was anastomosed to the fundus of the native bladder via the serosal layer, and the omentum and a double-J stent were placed in the decellularized bladder by no direct contact with the urine (Group A, n=8). In group B (n=8), the bladder was augmented applying the decellularized bladder that was in contact with the urine. After 6 months, the omentum was brought out of the neck of the engineered bladder and the anastomosis was opened. Biopsies were taken at 1, 3, and 9 months postoperatively. RESULTS: Cell removal with preservation of extracellular matrix structure was confirmed in decellularized bladders. Histological examination after 1 month demonstrated few cells at the border of the grafts. After 3 months, the region of the graft was indistinguishable from the natural bladder with continuity of transitional epithelium of natural bladder on the decellularized grafted scaffolds. The organization of muscle layers was similar to native bladder muscle layers after 9 months. IHC staining markers were highly expressed after 9 months. Interestingly, bladders had a high fibrosis grade in group B compared with hourglass technique. CONCLUSION: We confirmed that decellularized bladder may be a reliable scaffold and viable material for bladder augmentation.