Molecular engineering of the urethra for treatment of stress incontinence using a novel biomimetic aggrecan ex vivo and in vivo

摘要

Introduction: Stress urinary incontinence (SUI) is the involuntary leakage of urine through urethra during physical activity such as walking, exercising, lifting, and in severe cases, even laughing and coughing, affecting about 56% of postmenopausal women. SUI is associated with the decrease in volume and compliance of urethral tissue . Injecting bulking agents into urethral tissue is an underutilized treatment option for SUI, primarily because these treatments only last for up to 1 year due to migration of the bulking agents away from the urethra. Here we report our initial findings of using biomimetic aggrecan (BA) to molecularly engineer urethral tissue ex vivo and in vivo with the goal of increasing urethral compliance by bringing more hydration to the tissue because of the negatively charged BA molecules incorporated into the urethra extracellular matrix (ECM). Materials and Methods: BA was synthesized via a "grafting-to" method in which natural chondroitin sulfate bristles were coupled to functional groups on an enzymatically resistant polyacrylic acid core (250 kDa) in a 3D bottle brush configuration. Porcine urethras (Animal Technologies, Inc.) were purchased and injection and radial expansion techniques were developed to test the effect of BA on tissue compliance ex vivo. 25 gauge needles were utilized to inject porcine cadavers transurethrally with 0.5 ml BA solutions at 3 sites (120 ° apart), approximately 1-2 mm deep into tissue. BA concentrations of 25 and 50 mg/ml in 1XPBS were tested, and compared to non-injected control tissue (n=4). Radial expansion testing (RET) was performed by transurethrally inserting a balloon catheter into injected porcine urethra. In order to study integration of BA into the ECM, fluorescently-tagged BA was injected into porcine urethra and imaged using confocal microscopy. Micro-CT imaging was performed to study volume change of BA-injected porcine tissue. Biocompatibility of BA was tested in vivo by transurethrally injecting New Zealand White rabbits (n=6) with 0.5 ml of BA solution using a cystoscope (BA concentrations of 50 mg/ml and 200 mg/ml). Initial assessment of in vivo residence time was studied with fluorescently- labeled BA inejctions (figure 1). Urethras were harvested after 1 and 6 weeks, and histological analysis was performed. Results and Discussion: RET results show there is a statistically significant reduction in pressure of BA-injected samples; ～20% decrease (p＜0.05) and 35% decrease (p＜0.01) for 25 and 50 mg/ml BA injections, respectively indicating increased compliance was achieved, potentially allowing for better sealing of the urethra in incontinent women. An increase in tissue volume (bulking) was observed at the site of BA injection using Micro-CT imaging. The in vivo results demonstrate the host response of BA injections in the rabbit urethra at concentrations of 50 mg/ml and 200 mg/ml. Histological results show there is no morphological difference between BA injected groups and control groups (figure 2). Fluorescent imaging shows BA has infiltrated the urthral tissue and distributed throughout the tissue. Conclusion: BA mimics the structure and properties of natural aggrecan. It shows appropriate host response after injection into the rabbit urethra in vivo and has potential to reduce stiffness and increase the volume of tissue showing promise in reversing deteriorative tissue changes and may thus help incontinent patients.