Electrostimulation-Based Decellularized Matrix Bladder Patch Promotes Bladder Repair in Rats.

Bladder tissue engineering offers significant potential for repairing defects resulting from congenital and acquired conditions. However, the effectiveness of engineered grafts is often constrained by insufficient vascularization and neural regeneration. This study utilized four primary biomaterials─gelatin methacryloyl (GelMA), chitin nanocrystals (ChiNC), titanium carbide (MXene), and adipose-derived stem cells (ADSC)─to formulate two types of bioinks, GCM0.2 and GCM0.2-ADSC, in specified proportions. These bioinks were 3D printed onto bladder acellular matrix (BAM) patches to create BAM-GCM0.2 and BAM-GCM0.2-ADSC patches. The BAM-GCM0.2-ADSC patches underwent electrical stimulation to yield GCM0.2-ADSC-ES bladder patches. Employed for the repair of rat bladder defects, these patches were evaluated against a Control group, which underwent partial cystectomy followed by direct suturing. Our findings indicate that the inclusion of ADSC and electrical stimulation significantly enhances the regeneration of rat bladder smooth muscle (from [24.052 ± 2.782] % to [57.380 ± 4.017] %), blood vessels (from [5.326 ± 0.703] % to [12.723 ± 1.440] %), and nerves (from [0.227 ± 0.017] % to [1.369 ± 0.218] %). This research underscores the superior bladder repair capabilities of the GCM0.2-ADSC-ES patch and opens new pathways for bladder defect repair.

A bilayer bioengineered patch with sequential dual-growth factor release to promote vascularization in bladder reconstruction.

Bladder tissue engineering holds promise for addressing bladder defects resulting from congenital or acquired bladder diseases. However, inadequate vascularization significantly impacts the survival and function of engineered tissues after transplantation. Herein, a novel bilayer silk fibroin (BSF) scaffold was fabricated with the capability of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB) sequential release. The outer layer of the scaffold was composed of compact SF film with waterproofness to mimic the serosa of the bladder. The inner layer was constructed of porous SF matrix incorporated with SF microspheres (MS) loaded with VEGF and PDGF-BB. We found that the 5% (w/v) MS-incorporated scaffold exhibited a rapid release of VEGF, whereas the 0.2% (w/v) MS-incorporated scaffold demonstrated a slow and sustained release of PDGF-BB. The BSF scaffold exhibited good biocompatibility and promoted endothelial cell migration, tube formation and enhanced endothelial differentiation of adipose derived stem cells (ADSCs) in vitro. The BSF patch was constructed by seeding ADSCs on the BSF scaffold. After in vivo transplantation, not only could the BSF patch facilitate the regeneration of urothelium and smooth muscle, but more importantly, stimulate the regeneration of blood vessels. This study demonstrated that the BSF patch exhibited excellent vascularization capability in bladder reconstruction and offered a viable functional bioengineered patch for future clinical studies.

Application of antibody-conjugated small intestine submucosa to capture urine-derived stem cells for bladder repair in a rabbit model.

The need for bladder reconstruction and side effects of cystoplasty have spawned the demand for the development of alternative material substitutes. Biomaterials such as submucosa of small intestine (SIS) have been widely used as patches for bladder repair, but the outcomes are not fully satisfactory. To capture stem cells in situ has been considered as a promising strategy to speed up the process of re-cellularization and functionalization. In this study, we have developed an anti-CD29 antibody-conjugated SIS scaffold (AC-SIS) which is capable of specifically capturing urine-derived stem cells (USCs) in situ for tissue repair and regeneration. The scaffold has exhibited effective capture capacity and sound biocompatibility. In vivo experiment proved that the AC-SIS scaffold could promote rapid endothelium healing and smooth muscle regeneration. The endogenous stem cell capturing scaffolds has thereby provided a new revenue for developing effective and safer bladder patches.

3D Bioprinting of an In Vitro Model of a Biomimetic Urinary Bladder with a Contract-Release System.

The development of curative therapy for bladder dysfunction is usually hampered owing to the lack of reliable ex vivo human models that can mimic the complexity of the human bladder. To overcome this issue, 3D in vitro model systems offering unique opportunities to engineer realistic human tissues/organs have been developed. However, existing in vitro models still cannot entirely reflect the key structural and physiological characteristics of the native human bladder. In this study, we propose an in vitro model of the urinary bladder that can create 3D biomimetic tissue structures and dynamic microenvironments to replicate the smooth muscle functions of an actual human urinary bladder. In other words, the proposed biomimetic model system, developed using a 3D bioprinting approach, can recreate the physiological motion of the urinary bladder by incorporating decellularized extracellular matrix from the bladder tissue and introducing cyclic mechanical stimuli. The results showed that the developed bladder tissue models exhibited high cell viability and proliferation rate and promoted myogenic differentiation potential given dynamic mechanical cues. We envision the developed in vitro bladder mimicry model can serve as a research platform for fundamental studies on human disease modeling and pharmaceutical testing.

Biomaterials assisted reconstructive urology: The pursuit of an implantable bioengineered neo-urinary bladder.

Urinary bladder is a dynamic organ performing complex physiological activities. Together with ureters and urethra, it forms the lower urinary tract that facilitates urine collection, low-pressure storage, and volitional voiding. However, pathological disorders are often liable to cause irreversible damage and compromise the normal functionality of the bladder, necessitating surgical intervention for a reconstructive procedure. Non-urinary autologous grafts, primarily derived from gastrointestinal tract, have long been the gold standard in clinics to augment or to replace the diseased bladder tissue. Unfortunately, such treatment strategy is commonly associated with several clinical complications. In absence of an optimal autologous therapy, a biomaterial based bioengineered platform is an attractive prospect revolutionizing the modern urology. Predictably, extensive investigative research has been carried out in pursuit of better urological biomaterials, that overcome the limitations of conventional gastrointestinal graft. Against the above backdrop, this review aims to provide a comprehensive and one-stop update on different biomaterial-based strategies that have been proposed and explored over the past 60 years to restore the dynamic function of the otherwise dysfunctional bladder tissue. Broadly, two unique perspectives of bladder tissue engineering and total alloplastic bladder replacement are critically discussed in terms of their status and progress. While the former is pivoted on scaffold mediated regenerative medicine; in contrast, the latter is directed towards the development of a biostable bladder prosthesis. Together, these routes share a common aspiration of designing and creating a functional equivalent of the bladder wall, albeit, using fundamentally different aspects of biocompatibility and clinical needs. Therefore, an attempt has been made to systematically analyze and summarize the evolution of various classes as well as generations of polymeric biomaterials in urology. Considerable emphasis has been laid on explaining the bioengineering methodologies, pre-clinical and clinical outcomes. Some of the unaddressed challenges, including vascularization, innervation, hollow 3D prototype fabrication and urinary encrustation, have been highlighted that currently delay the successful commercial translation. More importantly, the rapidly evolving and expanding concepts of bioelectronic medicine are discussed to inspire future research efforts towards the further advancement of the field. At the closure, crucial insights are provided to forge the biomaterial assisted reconstruction as a long-term therapeutic strategy in urological practice for patients' care.

Poly-phosphate increases SMC differentiation of mesenchymal stem cells on PLGA-polyurethane nanofibrous scaffold.

The use of bioactive scaffolds in tissue engineering has a significant effect on the damaged tissue healing by an increase in speed and quality of the process. Herein, electrospinning was applied to fabricate composite nanofibrous scaffolds by Poly lactic-co-glycolic acid (PLGA) and Polyurethane (PU) with and without poly-phosphate (poly-P). Scaffolds were characterized morphologically by scanning electron microscope (SEM), and their biocompatibility was also investigated by SEM, protein adsorption, cell attachment and survival assays. The applicability of the scaffolds for bladder tissue engineering was also evaluated by culturing mesenchymal stem cells (MSCs) on the scaffolds and their differentiation into smooth muscle cell (SMC) was studied at the gene and protein levels. The results demonstrated that scaffold biocompatibility was increased significantly by loading poly-P. SMC related gene and protein expression level in MSCs cultured on poly-P-loaded scaffold was also increased significantly compared to those cells cultured on empty scaffold. It can be concluded that poly-P hasn't also increased scaffold biocompatibility, but also SMC differentiation potential of MSCs was also increased while cultured on the poly-P containing scaffold compared to the empty scaffold. Taken together, our study showed that PLGA-PU-poly-P alone and in combination with MSCs has a promising potential for support urinary bladder smooth muscle tissue engineering.

[Advance in functional bladder engineering].

Bladder has many important functions as a urine storage and voiding organ. Bladder injury caused by various pathological factors may need bladder reconstruction. Currently the standard procedure for bladder reconstruction is gastrointestinal replacement. However, due to the significant difference in their structure and function, intestinal segment replacement may lead to complications such as hematuria, dysuria, calculi and tumor. With the recent advance in tissue engineering and regenerative medicine, new techniques have emerged for the repair of bladder defects. This paper reviews the recent progress in three aspects of urinary bladder tissue engineering, i.e., seeding cells, scaffolds and growth factors.

Review of clinical experience on biomaterials and tissue engineering of urinary bladder.

PURPOSE: In recent pre-clinical studies, biomaterials and bladder tissue engineering have shown promising outcomes when addressing the need for bladder tissue replacement. To date, multiple clinical experiences have been reported. Herein, we aim to review and summarize the reported clinical experience of biomaterial usage and tissue engineering of the urinary bladder. METHODS: A systematic literature search was performed on Feb 2019 to identify clinical reports on biomaterials for urinary bladder replacement or augmentation and clinical experiences with bladder tissue engineering. We identified and reviewed human studies using biomaterials and tissue-engineered bladder as bladder substitutes or augmentation implants. The studies were then summarized for each respective procedure indication, technique, follow-up period, outcome, and important findings of the studies. RESULTS: An extensive literature search identified 25 studies of case reports and case series with a cumulative clinical experience of 222 patients. Various biomaterials and tissue-engineered bladder were used, including plastic/polyethylene mold, preserved dog bladder, gelatine sponge, Japanese paper with Nobecutane, lypholized human dura, bovine pericardium, amniotic membrane, small intestinal mucosa, and bladder tissue engineering with autologous cell-seeded biodegradable scaffolds. However, overall clinical experiences including the outcomes and safety reports were not satisfactory enough to replace enterocystoplasty. CONCLUSION: To date, several clinical experiences of biomaterials and tissue-engineered bladder have been reported; however, various studies have reported non-satisfactory outcomes. Further technological advancements and a better understanding is needed to advance bladder tissue engineering as a future promising management option for patients requiring bladder drainage.

Use of the extracellular matrix from the porcine esophagus as a graft for bladder enlargement.

INTRODUCTION: Some patients with diseases that involve increased bladder pressure or low-capacity bladders may need bladder enlargement surgery. In current techniques for bladder enlargement, autologous tissue such as small intestine or colon tissue is used to perform cystoplasties, which is far from ideal for these patients. In search of biomaterials with appropriate regeneration and safety profiles, tissue engineering has resulted in preclinical studies with acellular matrices in animal models that have yielded positive preliminary results with respect to the urothelial cell and smooth muscle repopulation; these studies have primarily been performed with matrices originating from the bladder or intestinal submucosa. OBJECTIVE: The aim of the study was to assess an extracellular matrix device derived from the porcine esophagus for augmentation cystoplasty in an animal model. STUDY DESIGN: Seven male Wistar rats weighing 357-390 g were subjected to augmentation cystoplasty with a circular segment of the acellular matrix from the porcine esophagus. Daily postoperative follow-up was performed with evaluation of changes in body weight, behavior, and wound status. RESULTS: During follow-up, there were no complications associated with the process. Three specimens were sacrificed at day 30, and three, at day 60. Necropsy was performed, with a description of the macroscopic findings and a morphological study. Epithelialization was observed, with different stages of mucosal development in all specimens analyzed. Repopulation of smooth muscle cells, mixed inflammatory infiltrate, and vascular neoformation were identified in the matrices. DISCUSSION: The urothelium and fibers of the smooth muscle were observed inside the implanted matrix. Additional investigations in larger animal models that allow urodynamic evaluation of the bladder with the matrix implanted are needed. However, to compare the results of this study model with those reported in the literature, a matrix derived from an organ different from the bladder was used because it could prevent the use of an intestinal segment in augmentation cystoplasty. CONCLUSION: The acellular porcine esophagus matrix offers positive results regarding the repopulation of the urothelium and smooth muscle when used in augmentation cystoplasty in a murine model.

Bladder smooth muscle cell differentiation of the human induced pluripotent stem cells on electrospun Poly(lactide-co-glycolide) nanofibrous structure.

Smooth muscle cell (SMC) regeneration plays an important role in retrieving the bladder-wall functionality and it can be achieved by a proper cell-co-polymer constructed by tissue engineering. Human induced pluripotent stem cells (iPSCs), which can be specifically prepared for the patient, was considered as cells in this study, and Poly(lactide-co-glycolide) (PLGA) as a most interesting polymer in biomedical applications was applied to the scaffold fabrication by electrospinning. After scaffold characterization, SMC differentiation potential of the human iPSCs was investigated while cultured on the PLGA nanofibrous scaffold by evaluation of the SMC related important gene and protein markers. Alpha-smooth muscle actin (ASMA), Smooth muscle 22 alpha (SM-22a) as two early SMC markers were significantly up regulated either two and three weeks after differentiation induction in human iPSCs cultured on PLGA compared to those cells cultured on the tissue culture polystyrene (TCPS). But Calponin-1, Caldesmon1 and myosin heavy chain (MHC) expression differences in human iPSCs cultured on PLGA and TCPS were significant only three weeks after differentiation induction based on its lately expression in the differentiation process. ASMA and MHC proteins were also considered for evaluation by immunocytochemistry on differentiated iPSCs whereas results showed higher expression of these proteins in stem cells grown on PLGA compared to the TCPS. According to the results, human iPSCs demonstrated a great SMC differentiation potential when grown on PLGA and it could be considered as a promising cell-co-polymer for use in bladder tissue engineering.

Biomimetic scaffold containing PVDF nanofibers with sustained TGF-ß release in combination with AT-MSCs for bladder tissue engineering.

Since the functional recovery of the reconstructed bladder is related to the bladder wall contraction and existing therapies do not respond to this, tissue engineering could be worth considered promising candidates for developing of conventional treatments in these kinds of ailments. Due to the low mechanical properties of natural scaffolds, biocompatible synthetic scaffolds can play a key role in the stem cells proliferation and differentiation and apply for many tissue-engineering applications. On the other hand, considering the low shelf life of TGFß, encapsulating this growth factor can help maintain its functionality throughout the study period. In this study, poly (vinylidene fluoride) (PVDF) nanofibrous scaffolds were fabricated through electrospinning method with or without chitosan nanoparticles loaded TGFß. All scaffolds characterized morphologically by using SEM, TGFß release profiling as well as biocompatibility by using SEM and MTT assays. Adipose tissue derived mesenchymal stem cells (AT-MSCs) was isolated and characterized immediately and the differentiation of SMC was investigated when cultured on the surface of the scaffolds and tissue culture polystyrene (TCPS) as a control of gene and protein expression levels. Fabricated scaffold possess smooth structure with nanoscale size and long time releasing of sustained profiles. MTT, qRT-PCR and immunocytochemistry results demonstrated that AT-MSCs proliferation rate and SMC differentiation potential were significantly increased when cultured on the PVDF-TGFß scaffold in comparison with PVDF and TCPS. According to the results, PVDF-TGFß as a bio-functional scaffold can provide greater treatment possibilities in bladder tissue engineering applications.

Bladder smooth muscle cells on electrospun poly(ε-caprolactone)/poly(l-lactic acid) scaffold promote bladder regeneration in a canine model.

Engineering of urinary bladder has been the focus of numerous studies in recent decade. Novel biomaterials, innovative fabrication methods and various modification processes of scaffolds are the critical issues to find supportive matrices. Supportive characteristics of electrospun PCL/PLLA nano-scaffold for bladder augmentation in canine model and the role of bladder cells in regeneration process were appraised. Electrospun PCL/PLLA was fabricated by co-electrospinning of PCL and PLLA. Bladder cells were isolated and transduced with lentiviral particles encoding eGFP and JRed proteins. Electrospun PCL/PLLA was seeded with different bladder cells individually or in co-culture condition. Cell-free and cell-seeded electrospun PCL/PLLA scaffolds (10cm2) were surgically implanted in bladders of eight female dogs for three months. To evaluate bladder regeneration, the dogs were sacrificed and their bladders were examined macroscopically and microscopically for presence of tracking proteins, expression of cell-specific markers and histological attributes of regenerated tissues. All animals survived the experiment with no complication. In smooth muscle transplanted group complete regeneration and covering of scaffold were observed. Other groups revealed partial regeneration. A well-developed layer of urothelium was formed in all groups in regenerated parts. Smooth muscle transplanted group showed the most developed muscle layer. Regenerated tissue demonstrated typical expression of cell-specific markers. No expression of eGFP and JRed was observed. Electrospun PCL/PLLA scaffold with proper handling, suture retention, nano-sized surface features, maintenance of normal phenotype of cells and minimal adverse effects in body can be a supportive substrate for bladder wall regeneration when seeded with bladder smooth muscle cells.

The synergistic effect of surface topography and sustained release of TGF-ß1 on myogenic differentiation of human mesenchymal stem cells.

A combination of topographical cues and controlled release of biochemical factors is a potential platform in controlling stem cells differentiation. In this study the synergistic effect of nanotopography and sustained release of biofunctional transforming growth factor beta 1 (TGF-ß1) on differentiation of human Wharton's Jelly-derived mesenchymal stem cell (hWJ-derived UC-MSCs) toward myogenic lineage was investigated. In order to achieve a sustained release of TGF-ß1, this factor was encapsulated within chitosan nanoparticles. Afterwards the aligned composite mats were fabricated using poly-ɛ-caprolacton (PCL) containing TGF-ß1-loaded chitosan nanoparticles and poly-L-lactic acid (PLLA). The nanofiber topography notably up-regulated the expressions of calponin1 and SM22α compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and sustained TGF-ß1release resulted in more significant enhancement of SMC marker, in particular smooth muscle α-actin (ASMA) expression, compared with bolus delivery despite lower amounts of TGF-ß1 (>10 times lower). Additionally, immunofluorescence staining showed that ASMA and desmin were expressed at higher intensity in cells exposed to controlled TGF-ß1 delivery rather than bolus delivery. These results demonstrated the importance of combined effect of topography and drug delivery in directing stem cell fate and the potential of such biofunctional scaffolds for cell transplantation applications in bladder tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1610-1621, 2016.

A dynamic distention protocol for whole-organ bladder decellularization: histological and biomechanical characterization of the acellular matrix.

A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation.

The promotion of functional urinary bladder regeneration using anti-inflammatory nanofibers.

Current attempts at tissue regeneration utilizing synthetic and decellularized biologic-based materials have typically been met in part by innate immune responses in the form of a robust inflammatory reaction at the site of implantation or grafting. This can ultimately lead to tissue fibrosis with direct negative impact on tissue growth, development, and function. In order to temper the innate inflammatory response, anti-inflammatory signals were incorporated through display on self-assembling peptide nanofibers to promote tissue healing and subsequent graft compliance throughout the regenerative process. Utilizing an established urinary bladder augmentation model, the highly pro-inflammatory biologic scaffold (decellularized small intestinal submucosa) was treated with anti-inflammatory peptide amphiphiles (AIF-PAs) or control peptide amphiphiles and used for augmentation. Significant regenerative advantages of the AIF-PAs were observed including potent angiogenic responses, limited tissue collagen accumulation, and the modulation of macrophage and neutrophil responses in regenerated bladder tissue. Upon further characterization, a reduction in the levels of M2 macrophages was observed, but not in M1 macrophages in control groups, while treatment groups exhibited decreased levels of M1 macrophages and stabilized levels of M2 macrophages. Pro-inflammatory cytokine production was decreased while anti-inflammatory cytokines were up-regulated in treatment groups. This resulted in far fewer incidences of tissue granuloma and bladder stone formation. Finally, functional urinary bladder testing revealed greater bladder compliance and similar capacities in groups treated with AIF-PAs. Data demonstrate that AIF-PAs can alleviate galvanic innate immune responses and provide a highly conducive regenerative milieu that may be applicable in a variety of clinical settings.

The use of bi-layer silk fibroin scaffolds and small intestinal submucosa matrices to support bladder tissue regeneration in a rat model of spinal cord injury.

Adverse side-effects associated with enterocystoplasty for neurogenic bladder reconstruction have spawned the need for the development of alternative graft substitutes. Bi-layer silk fibroin (SF) scaffolds and small intestinal submucosa (SIS) matrices were investigated for their ability to support bladder tissue regeneration and function in a rat model of spinal cord injury (SCI). Bladder augmentation was performed with each scaffold configuration in SCI animals for 10 wk of implantation and compared to non-augmented control groups (normal and SCI alone). Animals subjected to SCI alone exhibited a 72% survival rate (13/18) while SCI rats receiving SIS and bi-layer SF scaffolds displayed respective survival rates of 83% (10/12) and 75% (9/12) over the course of the study period. Histological (Masson's trichrome analysis) and immunohistochemical (IHC) evaluations demonstrated both implant groups supported de novo formation of smooth muscle layers with contractile protein expression [α-smooth muscle actin (α-SMA) and SM22α] as well as maturation of multi-layer urothelia expressing cytokeratin (CK) and uroplakin 3A proteins. Histomorphometric analysis revealed bi-layer SF and SIS scaffolds respectively reconstituted 64% and 56% of the level of α-SMA+ smooth muscle bundles present in SCI-alone controls, while similar degrees of CK+ urothelium across all experimental groups were detected. Parallel evaluations showed similar degrees of vascular area and synaptophysin+ boutons in all regenerated tissues compared to SCI-alone controls. In addition, improvements in certain urodynamic parameters in SCI animals, such as decreased peak intravesical pressure, following implantation with both matrix configurations were also observed. The data presented in this study detail the ability of acellular SIS and bi-layer SF scaffolds to support formation of innervated, vascularized smooth muscle and urothelial tissues in a neurogenic bladder model.

Increased porosity of electrospun hybrid scaffolds improved bladder tissue regeneration.

The object of this study was to investigate the role of scaffold porosity on tissue ingrowth using hybrid scaffolds consisting of bladder acellular matrix and electrospun poly (lactide-co-glycolide) (PLGA) microfibers that mimic the morphological characteristics of the bladder wall in vitro and in vivo. We compared single-spun (SS) PLGA scaffolds with more porous cospun (CS) scaffolds (PLGA and polyethylene glycol). Scaffolds were characterized by scanning electron microscopy. Bladder smooth muscle cells (SMCs) were seeded, and proliferation and histological assays were performed. Sixteen rats were subjected to augmentation cystoplasty with seeded SS or CS scaffolds, morphological, and histological studies were performed 2 and 4 weeks after implantation. The porosities of SS and CS scaffolds were 73.1 ± 2.9% and 80.9 ± 1.5%, respectively. The in vitro evaluation revealed significantly deeper cell migration into CS scaffolds. The in vivo evaluation showed significant shrinkage of SS scaffolds (p = 0.019). The histological analysis revealed a bladder wall-like structure with urothelial lining and SMC infiltration in both groups. The microvessel density was significantly increased in the CS scaffolds (p < 0.001). Increasing the porosity of electrospun hybrid scaffolds is an effective strategy to enhance cell proliferation and distribution in vitro and tissue ingrowth in vivo.