A bioactive injectable bulking material; a potential therapeutic approach for stress urinary incontinence
Introduction
Stress urinary incontinence (SUI) is uncontrolled leakage of urine caused by physical movements and activities such as exercising, sneezing or coughing. SUI can primarily result from weak muscle complex in the pelvic floor and/or of weak urethral sphincter muscle complex at the bladder neck. These conditions can be due to mechanical trauma during childbirth or hormonal changes. It causes a great deal of distress and embarrassment, as well as significant costs, towards both the patient and society [1]. A meta-analysis report estimated prevalence of 30% for urinary incontinence in women aged from 30 to 60 years, with approximately half of the cases related to SUI [2]. Physiotherapeutic exercises to strengthen the pelvic floor muscle complex, lifestyle modifications, loss of weight and drug therapy are recommended as first line-treatment options of SUI as they are usually associated with the least risk of complication [3]. However, if such non-invasive treatment options fail, second-line therapy is the surgical intervention. Retropubic and transobturator tension-free mid-urethral slings (MUS) are the most effective and popular procedures for the surgical treatment of female SUI [4]. These surgeries are currently considered the gold standard treatment for SUI, since they are related to high cure rates and effectiveness [5,6]. Nevertheless, several complications resulted from sling placement have been reported, including exposure and erosion of the material, permanent urinary retention, infection, bladder perforation, and persistent groin/suprapubic pain and dyspareunia in certain patients [7,8]. Bulking agent application is an alternative line of treatment in the management of SUI. The bulking agent is injected into the submucosal tissues of the urethra to increase urethral resistance; thus ameliorating SUI [9]. An ideal injectable bulking agent should therefore be tissue compatible, nonerosive, non-allergenic, and provoke minimal fibrosis at the injection site [10,11]. Commercially available bulking agents are made from different materials including carbon beads, calcium hydroxylapatite, glutaraldehyde crosslinked bovine collagen, polydimethylsiloxane particles, and hydrogel crosslinked with polyacrylamide hydrogel [9]. Therefore, urethral bulking agents would appear to have an important role in the management of SUI. However, in the last guidelines of the European Urology Association, it was suggested that bulking agents do not offer a permanent cure for patients with SUI due to discouraging long-term treatment results for SUI patients [12]. Therefore, bulking agent applications should be thought as an alternative treatment option for patients at high risk of potential post-operative complications [[13], [14], [15]].
Regenerative medicine might provide a solution to improve the outcome of bulking agents with the engineering of a combination therapy to produce a living tissue capable of providing long-term bulking function. Several studies have indicated that biological approaches including autologous fats, adipose-derived stem cells, and bioactive molecules (e.g. growth factors) might stimulate the regeneration of host tissues including nerve and muscle, and thus improving the urinary sphincter complex function [16,17]. Moreover, many studies have demonstrated the potential therapeutic use of several growth factors in bulking materials, such as insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF) [11,18,19]. Our previous studies indicated that our recombinant IGF-1 also improved smooth muscle regeneration in the rat bladder tissue [20,21]. Therefore, the incorporation of this recombinant IGF-1 within a bulking agent may add an advantage of treating the cause and not only reducing the signs and symptoms. The external (striated muscle) and internal (smooth muscle) urethral sphincter muscle function may be corrected with IGF-1 therapy. We have demonstrated in our previous study that tubular collagen scaffolds can induce regeneration of the urethra [22]. The triggering of neo-host regeneration at the injection site will improve the effectiveness of bulking agents. For the treatment of urinary incontinence, a persistent bulk is also a prerequisite and collagen durability at the injection site can be prolonged by crosslinking, causing changes in the arrangement of collagen fibrils. This results in a decrease in collagen absorption rates [23,24]. This approach has been employed using commercially available collagen based bulking agents to improve its outcome. Contigen™ (Bard Urology, Covington, Ga., USA) is bovine-derived collagen. Thus, skin testing is required, considering the risk of immune response [25]. In addition, it is rapidly absorbed and the effect is short lasting. After Contigen™ was retracted from the market, Permacol™ paste, which is also a collagen-based injectable material, has been used in the treatment of fecal incontinence [26]. Permacol™ is porcine collagen and its three dimensional orientation has not been distorted during manufacturing process [27]. Therefore, it has been suggested that no preoperative allergy testing is required [28]. In this paper, we aimed to combine the bulking capacity of Permacol™ with the muscle regenerative capacity of fibrin micro-beads conjugated with our recombinant IGF-1 (rIGF-1). rIGF-1 does not covalently bind to collagen and its covalent binding to fibrin overcomes the problem of burst release [21]. We have utilized a fibrin-binding variant of rIGF-1, namely α2PI1-8-MMP-IGF-1; the α2PI1-8 domain is covalently attached to fibrin during coagulation under the activity of factor XIIIa, and the MMP domain is a substrate for matrix metalloproteinases, allowing growth factor cleavage from the fibrin matrix under cell-derived enzymatic activity [29,30]. Fibrin micro-beads were produced using a droplet microfluidics platform, which has been also previously described by our group [31]. Fibrin micro-beads were then mixed with Permacol™ creating the injectable bulking agent. The biocompatibility of either Permacol™ alone or Permacol™ mixed with fibrin micro-beads was assessed by cell metabolic activity and Live/Dead assays. In vivo degradation and migration of the fibrin micro-beads was evaluated through the subcutaneous injection of fibrin micro-beads containing Permacol™ formulations in the mouse dorsal skin. The proposed bulking formulation will not only create an immediate bulking effect, but will further provide cell-mediated and sustained delivery of the growth factor at the injection site over a prolonged period, thus inducing functional tissue regeneration. Its potential long-term regenerative effect was evaluated by submucosal injections into the bladder wall of the rabbit, to examine the ingrowth of smooth muscle cells into the injected area. An ideal bulking agent providing long-term continence for the patient by regenerating functional smooth muscle tissue at the injection site has not been developed yet. By utilizing an already commercially approved bulking agent as a carrier biomaterial and combining it with a fibrin growth factor delivery platform allowing prolonged and local growth factor release, this bioactive injectable may provide an effective, long-lasting, and non-invasive therapy for patients suffering from SUI.
Section snippets
Cell culture
Human urinary tract smooth muscle cells (hSMCs) were extracted from human ureteral excision for duplicated ureters, and cultured as described previously [32]. In vitro experiments were performed using hSMCs between passage 5 and 7. hSMCs were cultured in minimum essential alpha medium (α-MEM), supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. All reagents and solvents were purchased from Gibco (Carlsbad, CA).
Preparation of injectable Permacol™ and fibrin micro-beads
Fibrin micro-beads, either containing 28 μg of rIGF-1 or
In vitro cell response to fibrin micro-beads mixed with Permacol™
In vitro culture of human smooth muscle cells (hSMCs) showed that the addition of either conjugated or non-conjugated fibrin micro-beads to Permacol™ did not change cell response. No significant difference in hSMCs metabolic activity was observed when Permacol™, fib_Permacol™, and fib_rIGF-1_Permacol™ were compared at day 3, 7 and 14 after cell seeding (Fig. 2A). Cell viability results were also in agreement with the observed cell metabolic activity. A high cell viability (mean of 82% for
Discussion
All currently available bulking agents used in the injection treatment of SUI seem to provide only a temporary treatment option to the patients. They require repeated injections in order to maintain their therapeutic efficacy. These repeated injections may result in scar tissue formation at the injection site, which is mechanically not compatible with the host tissue. The new generation of bulking materials should go beyond the reconstitution of continence only, but should induce permanent
Conclusion
In this study, we proposed a bioactive bulking agent that offers an improved therapeutic potential to regenerate sphincter muscle function, rather than treating only the symptoms of SUI. rIGF-1 conjugated fibrin micro-beads added to Permacol™ formed stable bulks and triggered smooth muscle tissue regeneration within the injected bulks 3 months post-surgery. Moreover, revascularization was also highest in bulk areas made of fib_rIGF-1_Permacol™, perhaps contributing to accelerated muscle
Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
Disclosure
The growth factor binding technology described in this paper is the submission of patents that have been filed by Eidgenössische Technische Hochschule Zürich, upon which J.A.H is named as one of the inventors. E.V., H.M.L, E.M.B., K.P., J.A.H, M.P.L., and P.F are named as inventors on a patent application filed by the Ecole Polytechnique Fédérale de Lausanne that covers the microfluidics technology described in this paper. The other authors do not have any conflict of interest to be declared.
Acknowledgements
The authors would like to thank the Histology and the Bioimaging and Optics Platform of EPFL. This work was supported by Gebert Rüf Stiftung grant GRS-072/16, CTI project-18011.1 and Bridge PoC (a joint programme by SNSF and Innosuisse) project- 20B1-1_173758. We thank Yves Bayon, Medtronic Switzerland, to kindly provide us with Permacol™.
References (62)
- et al.
Surgical management of stress urinary incontinence
Obstet. Gynaecol. Reprod. Med.
(2016) - et al.
Definition of overactive bladder and epidemiology of urinary incontinence
Urology
(1997) - et al.
TVT-O for the treatment of pure urodynamic stress incontinence: efficacy, adverse effects, and prognostic factors at 5-year follow-up
Eur. Urol.
(2013) - et al.
Tension-free vaginal tape for the treatment of urodynamic stress incontinence: efficacy and adverse effects at 10-year follow-up
Eur. Urol.
(2012) - et al.
A comprehensive review of suburethral sling procedure complications
J. Minim. Invasive Gynecol.
(2008) - et al.
Urethral bulking agents versus other surgical procedures for the treatment of female stress urinary incontinence: a systematic review and meta-analysis
Eur. J. Obstet. Gynecol. Reprod. Biol.
(2015) - et al.
Stress incontinence injection therapy: what is best for our patients?
Eur. Urol.
(2005) - et al.
Post radical hysterectomy urinary incontinence: a prospective study of transurethral bulking agents injection
Gynecol. Oncol.
(2009) - et al.
Sphincter incontinence: is regenerative medicine the best alternative to restore urinary or anal sphincter function
Int. J. Biochem. Cell Biol.
(2007) - et al.
Periurethral injection of autologous adipose-derived stem cells with controlled-release nerve growth factor for the treatment of stress urinary incontinence in a rat model
Eur. Urol.
(2011)
Engineered insulin-like growth factor-1 for improved smooth muscle regeneration
Biomaterials
IGF-1-containing multi-layered collagen-fibrin hybrid scaffolds for bladder tissue engineering
Acta Biomater.
Engineered acellular collagen scaffold for endogenous cell guidance, a novel approach in urethral regeneration
Acta Biomater.
Local tissue reaction to the suburethral injection of glutaraldehyde cross-linked bovine collagen in humans
J. Urol.
Biocompatibility assessment of synthetic sling materials for female stress urinary incontinence
J. Urol.
Particle migration after transurethral injection of carbon coated beads for stress urinary incontinence
J. Urol.
Stress urinary incontinence animal models as a tool to study cell-based regenerative therapies targeting the urethral sphincter
Adv. Drug Deliv. Rev.
Regenerative medicine therapies for stress urinary incontinence
J. Urol.
Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase
J. Vasc. Surg.
Incorporation of fibrin into a collagen–glycosaminoglycan matrix results in a scaffold with improved mechanical properties and enhanced capacity to resist cell-mediated contraction
Acta Biomater.
Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells
Am. J. Pathol.
Therapeutic effects of IGF-1 on stress urinary incontinence in rats with simulated childbirth trauma
J. Urol.
Insulin-like growth factor-1 contributes to neovascularization in age-related macular degeneration
Biochem. Biophys. Res. Commun.
Skeletal myogenic differentiation of urine-derived stem cells and angiogenesis using microbeads loaded with growth factors
Biomaterials
The treatment of female stress urinary incontinence: an evidenced-based review
Open Access J. Urol.
Surgical treatment for female stress urinary incontinence: what is the gold-standard procedure?
Int. Urogynecol. J. Pelvic Floor Dysfunct.
Analysis of patient and technical factors associated with midurethral sling mesh exposure and perforation
Int. J. Urol.
Efficacy of the pubovaginal rectus fascia sling in the management of female patients suffering from complex intrinsic sphincteric deficiency (type III stress urinary incontinence)
Evid. Based Wom. Health J.
Bioactive porous beads as an injectable urethral bulking agent: their in vitro evaluation on smooth muscle cell differentiation
Tissue Eng. A
Guidelines on Urinary Incontinence European Urology
Urethral bulking agents: techniques and outcomes
Curr. Urol. Rep.
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