Alkaloids from Phaeanthus vietnamensis with inhibitory effect on nitric oxide production lipopolysaccharide-stimulated in RAW264.7 macrophages.

The chemical study of the acidic extract of Phaeanthus vietnamensis leaves led to the isolation of one new alkaloid, vietnamine A (1) and eight known alkaloids (R,S)-2N-norberbamunine (2), grisabine (3), 1S,1'R,O,O'-dimethylgrisabine (4), dauricine (5), neothalibrine (6), vietnamine (7), xylopine (8), and argentinine (9) by NMR and MS and comparing with the data reported in the literature. Compounds 1-9 were evaluated for inhibitory NO production in RAW 264.7 macrophages, LPS-stimulated. Compounds 1-3 significantly inhibited on NO production with the IC50 values of 6.8 ± 0.9, 9.8 ± 1.0, and 7.1 ± 0.4 µg/ml, respectively.

Prevalence of high-risk bladder categorization with prenatal and postnatal myelomeningocele repair types.

INTRODUCTION: Urologic substudies of prenatal myelomeningocele (MMC) closure have focused primarily on continence without significant clinical benefit. Fetoscopic MMC repair (FMR) is a newer form of prenatal intervention and touts added benefits to the mother, but urological outcomes have yet to be analyzed. We set out to focus on bladder safety rather than continence and examined bladder outcomes with different prenatal MMC repairs (FMR and prenatal open [POMR]) and compared bladder-risk-categorization to traditional postnatal repair (PSTNR). METHODS: An IRB-approved retrospective analysis of all patients undergoing all forms of MMC repairs with inclusion and exclusion criteria based on the MOMS trial was performed. Bladder safety assessment required initial urodynamic studies (UDS), renal bladder ultrasound (RBUS), and/or voiding cystourethrogram (VCUG) within the 1st year of life. Follow-up analyses within the cohorts required follow-up studies within 18 months after initial evaluations. Outcomes assessed included bladder-risk-categorization based on the CDC UMPIRE study (high, intermediate, and safe), hydronephrosis (HN), and vesicoureteral reflux (VUR). A single reader evaluated each UDS. RESULTS: Initial UDS in 93 patients showed that the prevalence of high-risk bladders were 35% FMR versus 36% PSTNR and 60% POMR. Follow-up UDS showed only 8% of FMR were high-risk compared to 35% POMR and 36% PSTNR. Change from initial to follow-up bladder-risk-category did not reach significance (p = .0659); however, 10% PSTNR worsened to high-risk on follow-up, compared to none in either prenatal group. Subanalysis of follow-up UDS between the prenatal cohorts also was not significant (p = .055). Only 8% of FMR worsened or stayed high-risk compared to 35% with POMR (p = .1). HN was significantly different at initial and subsequent follow up between the groups with the least in the FMR group. CONCLUSIONS: Early outcome UDS analyses demonstrated lower incidence of high-risk bladders in FMR patients with a trend toward clinically significant improvement compared to POMR in regard to all evaluated metrics. Larger, prospective, confirmatory studies are needed to further evaluate the potential benefits on FMR on bladder safety and health.

Open versus robot-assisted laparoscopic ureteral reimplantation: Hospital charges analysis and outcomes at a single institution.

PURPOSE: Robot-assisted laparoscopic extravesical ureteral reimplantation has previously been described as a viable minimally invasive option to open surgery. However, concerns for robotic surgery have been raised owing to assumed higher costs and heterogeneous clinical outcomes. We hypothesized that similar hospital charges and clinical outcomes occur when comparing open and robotic cases in matched cohorts. MATERIALS AND METHODS: Open and robotic reimplantation cases from 2013 to 2015 for primary vesicoureteral reflux were matched by age using 1:1 nearest neighbor matching. The matched cohorts were analyzed and compared for their direct itemized hospital charges per surgical case, complications, and clinical outcomes. RESULTS: There were 38 patients in each group after age-matching the 135 patients. Operating room charges were higher for the robotic group compared to the open group (p=0.002), whereas pharmacy and laboratory costs were lower for the robotic group. However, there were no significant differences in total overall charges between the open and robotic groups with cystoscopy or without cystoscopy (p=0.345, p=0.533), since the median hospital stay length was shorter for the robotic group (p<0.001). Clinical success rates were identical for the two groups (open 94.8% vs robotic 94.8%). There were also no significant differences in number of complications between the two cohorts. CONCLUSIONS: This is the first age-matched study comparing hospital charges and clinical outcomes of pediatric open and robotic reimplantation. While operating room charges were higher for the robotic cohort, lower hospitalization charges led to comparable overall hospital charges, as well as equivalent clinical outcomes for both cohorts. LEVEL OF STUDY: Level III (Retrospective comparative study) TYPE OF STUDY: Retrospective Study.

Evaluating Clinical Outcomes of an Advanced Practice Provider-Led Newborn Circumcision Clinic.

OBJECTIVE: To determine the safety and efficacy of advanced practice provider (APP)-performed newborn circumcisions (NBCs), we reviewed outcomes of NBCs performed by pediatric urologists and APPs. We hypothesize comparable clinical outcomes between the groups. METHODS: All urology performed NBCs during a 5-year period were reviewed, including time surrounding implementation of the APP-led clinic. Return to emergency department (ED) rates, return to operating room (OR) rates, and intraprocedure bleeding requiring intervention were reviewed. Fisher exact and Mann-Whitney testing were utilized. RESULTS: There were no statistically significant differences in rates of intraprocedure bleeding, return to ED in 30days, return to OR for revision or other related penile surgery, or the overall number of patients with complications between the groups. Thirteen patients had complications in the APP cohort, compared to 8 in the urologist cohort. There was a difference in age and weight, with urologists performing NBCs on older and heavier patients. There was no difference in clinical outcomes between children over and under 10 pounds (4.5 kg). There was a significant difference in the need for revision circumcision when comparing children older vs younger than 30days (1.9% vs 0%, P = 0.034). CONCLUSION: An APP-led NBC clinic is both safe and feasible. The widely used age and weight cutoffs for NBC need to be further evaluated, as there was no significant difference in clinical outcomes. This practice design provides pediatric urologists more time to focus on the most complex patients, both in the clinic and OR.

Baseline Urinary Tract Imaging in Infants Enrolled in the UMPIRE Protocol for Children with Spina Bifida.

PURPOSE: The lifetime risk of renal damage in children with spina bifida is high but only limited baseline imaging data are available for this population. We evaluated a large prospective cohort of infants with spina bifida to define their baseline imaging characteristics. MATERIALS AND METHODS: The UMPIRE Protocol for Young Children with Spina Bifida is an iterative quality improvement protocol that follows a cohort of newborns at 9 United States centers. Using descriptive statistics, we report the initial baseline imaging characteristics, specifically regarding renal bladder ultrasound, cystogram and dimercaptosuccinic acid nuclear medicine scan. RESULTS: Data on 193 infants from 2015 to 2018 were analyzed. Renal-bladder ultrasound was normal in 55.9% of infants, while 40.4% had Society for Fetal Urology grade 1 to 2 hydronephrosis in at least 1 kidney, 3.7% had grade 3 to 4 hydronephrosis in either kidney and 21.8% had grade 1 or higher bilateral hydronephrosis. There was no vesicoureteral reflux in 84.6% of infants. A third of enrolled infants underwent dimercaptosuccinic acid nuclear medicine renal scan, of whom 92.4% had no renal defects and 93.9% had a difference in differential function of less than 15%. CONCLUSIONS: The majority of infants born with spina bifida have normal baseline imaging characteristics and normal urinary tract anatomy at birth. This proactive protocol offers careful scheduled surveillance of the urinary tract with the goal of lifelong maintenance of normal renal function and healthy genitourinary development.

Complex Robotic Lower Urinary Tract Surgery in Patients with History of Open Surgery.

PURPOSE: We describe our experience with robot-assisted complex lower urinary tract reconstruction in patients with a history of open abdominal surgery. MATERIALS AND METHODS: Patients with any previous open abdominal surgery undergoing robot-assisted complex lower urinary tract reconstruction were included. Complex lower urinary tract reconstruction was defined as bladder neck reconstruction or continent catheterizable conduits or both, redo surgery at the bladder neck for persistent incontinence or any of these procedures with creation of a Malone antegrade continence enema. Ureteral and renal surgeries were excluded. Patient demographics, surgery performed, operative techniques, operative times and outcomes were assessed. RESULTS: A total of 36 patients met inclusion criteria, of whom 21 had undergone multiple laparotomies for ventriculoperitoneal shunt revision, 14 had undergone laparotomy with other adjunct procedures and 1 had undergone laparotomy with colostomy. No access injury occurred and there were 5 conversions. Mean operative time was 8.2 hours (range 4 to 12) and mean length of hospital stay was 74.9 hours (23 to 216). The first 18 cases took longer than the last 18 cases (mean 9.1 vs 7.5 hours, p = 0.002). Patients with multiple ventriculoperitoneal shunt revisions had higher conversion rates (p = 0.01) and longer mean operative times (p = 0.002). Patients with a history of multiple ventriculoperitoneal shunt revisions also had longer hospital stays (p = 0.02). CONCLUSIONS: Robot-assisted complex lower urinary tract reconstruction in patients with previous open abdominal surgery is safe and feasible. Longer operative times should be expected early in the experience of a surgeon. Patients with multiple prior ventriculoperitoneal shunt revisions had higher conversion rates and longer operative times compared to those with other indications for prior surgery.

Surgical interventions and anesthesia in the 1st year of life for lower urinary tract obstruction.

BACKGROUND: Patients with a prenatal diagnosis of lower urinary tract obstruction (LUTO) may undergo prenatal interventions, such as vesicoamniotic shunt (VAS) placement, as a temporary solution for relieving urinary tract obstruction. A recent FDA communication has raised awareness of the potential neurocognitive adverse effects of anesthesia in children. We hypothesized as to whether a prenatal LUTO staging system was predictive of the number of anesthesia events for prenatally diagnosed LUTO patients. METHODS: We retrospectively reviewed the prenatal and postnatal clinical records for patients with prenatally diagnosed LUTO from 2012 to 2015. Patients were stratified by prenatal VAS status and by LUTO disease severity according to Ruano et al. (Ultrasound Obstet Gynecol. 2016). RESULTS: 31 patients were identified with a prenatal LUTO diagnosis, and postnatal records were available for 21 patients (seven patients in each stage). When combining prenatal and postnatal anesthesia, there was a significant difference in the number of anesthesia encounters by stage (1.6, 3.7, and 6.7 for Stage I, II, and III respectively, p = .034). Upon univariate analysis, higher gestational age (GA) at birth was associated with a decreased number of anesthesia events in the first year (p = .031). CONCLUSIONS: The majority of infants with prenatally diagnosed LUTO will undergo postnatal procedures with general anesthesia exposure in the first year of life. Patients with higher prenatal LUTO severity experienced a higher number of both prenatal and postnatal anesthesia encounters. In addition, higher GA at birth was associated with fewer anesthesia encounters in the first year. LEVEL OF EVIDENCE: This is a prognostic study with Level IV evidence.

Evaluating Outcomes of Differential Surgical Management of Nonfunctioning Upper Pole Renal Moieties in Duplex Collecting Systems.

OBJECTIVE: To evaluate the management and clinical outcomes of nonfunctioning upper pole moieties treated with either upper pole heminephrectomy or upper pole preservation with lower ureteral reconstruction at a single tertiary institution. METHODS: After Institutional Review Board (IRB) approval, patients with duplicated systems undergoing upper pole heminephrectomy, ureteroureterostomy, or common sheath ureteral reimplantation from 2012-2017 were identified. Only patients with a nonfunctioning upper pole moiety on ultrasound or renal scan were included. Patients undergoing upper pole heminephrectomy were compared to those undergoing upper pole preservation with respect to demographics, anatomic variations preoperatively, and postoperative outcomes. RESULTS: Twenty-seven (57%) patients underwent upper pole preservation with lower ureteral reconstruction; 20 (43%) patients underwent upper pole heminephrectomy. Patients undergoing lower ureteral reconstruction were older (1.63 vs 2.76 years, P = .018) and more commonly presented with lower pole vesicoureteral reflux (67% vs 25%, P = .008). No significant difference in postoperative complications was seen between the two groups. After ureteroureterostomy, one patient developed new onset symptomatic reflux to the upper pole requiring intravesical reimplantation. In the heminephrectomy group, 4 of 11 patients with ureteroceles had ureterocelectomy with concomitant lower pole reimplantation. After heminephrectomy, two additional patients required further interventions: ureterocele excision and transurethral polyp excision. CONCLUSION: For patients with nonfunctional upper poles, lower tract reconstruction is a safe alternative to upper pole heminephrectomy. No significant difference in outcomes was seen. Considering that nearly 1 of 3 of patients with upper pole heminephrectomy required additional lower urinary tract procedures, pursuing upper pole preservation with lower urinary tract reconstruction may be favorable.

Evaluating Natural History and Follow Up Strategies for Non-obstructive Urolithiasis in Pediatric Population.

Objective: While small non-obstructive stones in the adult population are usually observed with minimal follow-up, the same guidelines for management in the pediatric population have not been well-studied. We evaluate the clinical outcomes of small non-obstructing kidney stones in the pediatric population to better define the natural history of the disease. Methods: In this IRB-approved retrospective study, patients with a diagnosis of kidney stones from January 2011 to March 2017 were identified using ICD9 and ICD10 codes. Patients with ureteral stones, obstruction, or stones >5 mm in size were excluded. Patients with no follow-up after initial imaging were also excluded. Patients with a history of stones or prior stone interventions were included in our population. Frequency of follow-up ultrasounds while on observation were noted and any ER visits, stone passage episodes, infections, and surgical interventions were documented. Results: Over the 6-year study period, 106 patients with non-obstructing kidney stones were identified. The average age at diagnosis was 12.5 years and the average stone size was 3.6 mm. Average follow-up was 17 months. About half of the patients had spontaneous passage of stones (54/106) at an average time of 13 months after diagnosis. Stone location did not correlate with spontaneous passage rates. Only 6/106 (5.7%) patients required stone surgery with ureteroscopy and/or PCNL at an average time of 12 months after initial diagnosis. The indication for surgery in all 6 cases was pain. 17/106 (16%) patients developed febrile UTIs and a total of 43 ER visits for stone-related issues were noted, but no patients required urgent intervention for an infected obstructing stone. Median interval for follow-up was every 6 months with renal ultrasounds, which then was prolonged to annual follow up in most cases. Conclusions: The observation of pediatric patients with small non-obstructing stones is safe with no episodes of acute obstructive pyelonephritis occurring in these patients. The sole indication for intervention in our patient population was pain, which suggests that routine follow-up ultrasounds may not be necessary for the follow-up of pediatric non-obstructive renal stones ≤5 mm in size.

Vesicoamniotic Shunting Improves Outcomes in a Subset of Prune Belly Syndrome Patients at a Single Tertiary Center.

Objective: Review outcomes of Prune Belly Syndrome (PBS) with the hypothesis that contemporary management improves mortality. Methods: A retrospective chart review of inpatient and outpatient PBS patients referred between 2000 and 2018 was conducted to assess outcomes at our institution. Data collected included age at diagnosis, concomitant medical conditions, imaging, operative management, length of follow-up, and renal function. Results: Forty-five PBS patients presented during these 18 years. Prenatal diagnoses were made in 17 (39%); 65% of these patients underwent prenatal intervention. The remaining patients were diagnosed in the infant period (20, 44%) or after 1 year of age (8, 18%). Twelve patients died from cardiopulmonary complications in the neonatal period; the neonatal mortality rate was 27%. The mean follow-up among patients surviving the neonatal period was 84 months. Forty-two patients had at least one renal ultrasound (RUS); of the 30 patients with NICU RUSs, 26 (89%) had hydronephrosis and/or ureterectasis. Of the 39 patients who underwent voiding cystourethrogram (VCUG), 28 (62%) demonstrated VUR. Fifty-nine percent had respiratory distress. Nine patients (20%) were oxygen-dependent by completion of follow up. Thirty-eight patients (84%) had other congenital malformations including genitourinary (GU) 67%, gastrointestinal (GI) 52%, and cardiac 48%. Sixteen patients (36%) had chronic kidney disease (CKD) of at least stage 3; three patients (7%) had received renal transplants. Eighty-four percent of patients had at least one surgery (mean 3.4, range 0-6). The most common was orchiopexy (71%). The next most common surgeries were vesicostomy (39%), ureteral reimplants (32%), abdominoplasty (29%), nephrectomy (25%), and appendicovesicostomy (21%). After stratifying patients according to Woodard classification, a trend for 12% improvement in mortality after VAS was noted in the Woodard Classification 1 cohort. Conclusions: PBS patients frequently have multiple congenital anomalies. Pulmonary complications are prevalent in the neonate while CKD (36%) is prevalent during late childhood. The risk of CKD increased significantly with the presence of other congenital anomalies in our cohort. Mortality in childhood is most common in infancy and may be as low as 27%. Contemporary management of PBS, including prenatal interventions, reduced the neonatal mortality rate in a subset of our cohort.

Early postnatal bladder function in fetoscopic myelomeningocele repair patients.

PURPOSE: Prenatal repair of myelomeningocele (MMC) via hysterotomy has demonstrated neurosurgical and motor benefits, when compared to postnatal repairs. Urologic benefits, however, remain to be seen. The purpose of this study was to review early postnatal bladder function in patients undergoing a novel endoscopic approach for MMC repair using an exteriorized uterus. METHODS: A prospective urologic assessment of patients undergoing fetoscopic MMC repair and receiving subsequent care at our facility, was performed. Patients were managed and urodynamic studies risk-stratified according to the Centers for Disease Control and Prevention Urologic and Renal Protocol for the Newborn and Young Child with Spina Bifida. RESULTS: Fetoscopic MMC repair was performed in 14 patients. No patients had hydronephrosis or bladder thickening at birth. Detrusor overactivity was observed in nine (64.3%) patients. Impaired compliance was seen in eight (57.1%) patients. No patients had a detrusor leak point pressure of > 40 cm H2O or evidence of detrusor sphincter dyssynergia. Three (21.4%) patients had vesicoureteral reflux, seven (50.0%) had an open bladder neck, and none had trabeculated bladders. CONCLUSION: In this early experience with fetoscopic MMC repair, postnatal bladder function does not appear to be any worse than that of previously reported prenatal or postnatal closures.

Urologic Outcomes of Children With Hemorrhagic Cystitis After Bone Marrow Transplant at a Single Institution.

OBJECTIVE: To analyze clinical outcomes and the risk factors associated with genitourinary (GU) morbidity and mortality in children who present with hemorrhagic cystitis (HC) after bone marrow transplant (BMT). METHODS: A retrospective chart review of patients with HC who had undergone BMT at a single pediatric hospital from 2008 to 2015 was conducted. Demographic data, severity of hematuria, HC management, and mortality were analyzed. Bivariate analysis and binary logistic regression were performed to identify risk factors. RESULTS: Out of 43 patients who met inclusion criteria, 67.4% were male with a median age at BMT of 10.2 years (interquartile range 5.8-14.6). Percutaneous nephrostomy catheters were inserted in 5 patients for urinary diversion. All-cause mortality was 32.6% (N = 14). Intravesical retroviral therapy (P <.001), HC grade (P <.001), total Foley time (P <.001), total gross hematuria time (P <.001), total days hospitalized (P = .012), and days to most improved hematuria (P = .032) were associated with significant GU morbidity on bivariate analysis. On multivariable analysis, days to most improved hematuria was associated with significant GU morbidity odds ratio of 1.177 (1.006-1.376) (P = .042). Status of percutaneous nephrostomy was not associated with increased mortality (P = .472); however, in the multivariate model, BK viremia (P = .023), need for renal dialysis (P = .003), and presence of Foley catheter (P = .005) were associated with increased mortality. CONCLUSION: Children with HC after BMT fall in a very high-risk category with high mortality and significant GU morbidity. The presence of a Foley catheter, need for dialysis, and BK viremia are associated with increased mortality.

Evidence-Based Management of Patients with 45,X/46,XY Gonadal Dysgenesis and Male Sex Assignment: from Infancy to Adulthood.

45,X/46,XY gonadal dysgenesis is a disorder of sexual differentiation with a wide clinical presentation, ranging from Turner-like females to individuals with genital ambiguity to azoospermic but otherwise normal-appearing males. Hence, patients can be assigned female or male sex. Female patients are managed according to the Turner Syndrome Guidelines, whereas males are managed on a case-by-case basis. Male patients present with multiple medical challenges: undervirilization, hypogonadism, gonadoblastoma risk, and short stature. Many require surgeries and hormonal treatments that are time-sensitive and irreversible. Nonetheless, these therapeutic decisions are made without evidence-based guidelines. This review describes the medical concerns and possible interventions in male patients with 45,X/46,XY dysgenesis for each stage of development. Interventions should be addressed within a patient-centered framework by a multidisciplinary team and after thorough discussion with the family. We use the GRADE system to appraise the existing evidence and provide recommendations based on the available evidence.

Vacuum-assisted Closure of a Vesicocutaneous Fistula in a Pediatric Patient After Bladder Cystoplasty.

A vesicocutaneous fistula is an abnormal communication from the bladder to the skin (Pritts et al, 2001). Recently, wound vacuum-assisted closure (VAC) has been used to facilitate fistula closure. There are no reports of using VAC to help fistula closure in the pediatric population. We present a case of an adolescent patient who develops a vesicocutaneous fistula after bladder augment cystoplasty and was treated with VAC only.

Inosine Improves Neurogenic Detrusor Overactivity following Spinal Cord Injury.

Neurogenic detrusor overactivity and the associated loss of bladder control are among the most challenging complications of spinal cord injury (SCI). Anticholinergic agents are the mainstay for medical treatment of detrusor overactivity. However, their use is limited by significant side effects such that a search for new treatments is warranted. Inosine is a naturally occurring purine nucleoside with neuroprotective, neurotrophic and antioxidant effects that is known to improve motor function in preclinical models of SCI. However, its effect on lower urinary tract function has not been determined. The objectives of this study were to determine the effect of systemic administration of inosine on voiding function following SCI and to delineate potential mechanisms of action. Sprague-Dawley rats underwent complete spinal cord transection, or cord compression by application of an aneurysm clip at T8 for 30 sec. Inosine (225 mg/kg) or vehicle was administered daily via intraperitoneal injection either immediately after injury or after a delay of 8 wk. At the end of treatment, voiding behavior was assessed by cystometry. Levels of synaptophysin (SYP), neurofilament 200 (NF200) and TRPV1 in bladder tissues were measured by immunofluorescence imaging. Inosine administration decreased overactivity in both SCI models, with a significant decrease in the frequency of spontaneous non-voiding contractions during filling, compared to vehicle-treated SCI rats (p<0.05), including under conditions of delayed treatment. Immunofluorescence staining demonstrated increased levels of the pan-neuronal marker SYP and the Adelta fiber marker NF200, but decreased staining for the C-fiber marker, TRPV1 in bladder tissues from inosine-treated rats compared to those from vehicle-treated animals, including after delayed treatment. These findings demonstrate that inosine prevents the development of detrusor overactivity and attenuates existing overactivity following SCI, and may achieve its effects through modulation of sensory neurotransmission.

Acellular bi-layer silk fibroin scaffolds support tissue regeneration in a rabbit model of onlay urethroplasty.

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a rabbit model of urethra repair. A bi-layer silk fibroin matrix was fabricated by a solvent-casting/salt leaching process in combination with silk fibroin film casting to generate porous foams buttressed by homogeneous silk fibroin films. Ventral onlay urethroplasty was performed with silk fibroin grafts (Group 1, N = 4) (Width × Length, 1 × 2 cm(2)) in adult male rabbits for 3 m of implantation. Parallel control groups consisted of animals receiving small intestinal submucosa (SIS) implants (Group 2, N = 4) or urethrotomy alone (Group 3, N = 3). Animals in all groups exhibited 100% survival prior to scheduled euthanasia and achieved voluntary voiding following 7 d of initial catheterization. Retrograde urethrography of each implant group at 3 m post-op revealed wide urethral calibers and preservation of organ continuity similar to pre-operative and urethrotomy controls with no evidence of contrast extravasation, strictures, fistulas, or stone formation. Histological (hematoxylin and eosin and Masson's trichrome), immunohistochemical, and histomorphometric analyses demonstrated that both silk fibroin and SIS scaffolds promoted similar extents of smooth muscle and epithelial tissue regeneration throughout the original defect sites with prominent contractile protein (α-smooth muscle actin and SM22α) and cytokeratin expression, respectively. De novo innervation and vascularization were also evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Following 3 m post-op, minimal acute inflammatory reactions were elicited by silk fibroin scaffolds characterized by the presence of eosinophil granulocytes while SIS matrices promoted chronic inflammatory responses indicated by mobilization of mononuclear cell infiltrates. The results of this study demonstrate that bi-layer silk fibroin scaffolds represent promising biomaterials for onlay urethroplasty, capable of promoting similar degrees of tissue regeneration in comparison to conventional SIS scaffolds, but with reduced immunogenicity.

Bladder tissue regeneration using acellular bi-layer silk scaffolds in a large animal model of augmentation cystoplasty.

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm(2)) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth.In addition, animals augmented with both matrix configurations displayed increases in bladder compliance over pre-operative levels(Group 1: 357%; Group 2: 338%) similar to growth-related elevations observed in non-surgical controls (354%) [corrected]. Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,ß-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair.

The performance of silk scaffolds in a rat model of augmentation cystoplasty.

The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5-50 µm) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ~400 µm) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ≥57% and stone diameters of 3-4 mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2 mm), similar to the levels observed in controls (13%, 2 mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures.

Evaluation of biomaterials for bladder augmentation using cystometric analyses in various rodent models.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity(1). Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures(2). Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails(3). However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy(4,5). Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic(6,7). However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis(8), graft contracture(9), and calcification(10), thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed. In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.(11,12) Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders(11,12). Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model(11,12). These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.