Advances Toward Tissue Engineering for the Treatment of Stress Urinary Incontinence

Treatment Update

TREATMENT UPDATE Advances Toward Tissue Engineering for the Treatment of Stress Urinary Incontinence Ron Jankowski, PhD, Ryan Pruchnic, MS, Michael Hiles, PhD, Michael B. Chancellor, MD Department of Urology, University of Pittsburgh, Pittsburgh, PA Suburethral pubovaginal sling placement is a common surgical procedure for the treatment of stress urinary incontinence. A wide variety of graft materials is available, each associated with inherent desirable and undesirable characteristics and complications. In this article, we discuss the rationale for and application of small intestinal submucosa (SIS) in lower urinary tract tissue engineering, with emphasis on the use of SIS as a suitable and biologically compatible sling material. In addition, we discuss exciting research regarding the engineering of true functional sphincter reconstruction using this biologic scaffold and pre-seeded muscle cells. [Rev Urol. 2004;6(2):51-57] © 2004 MedReviews, LLC Key words: Pubovaginal sling • Small intestinal submucosa • Stress urinary incontinence • Xenografts S tress urinary incontinence (SUI) is a major urologic health problem affecting approximately 25 million American women,1 and the number of patients with SUI will rise dramatically as the baby-boomer generation continues to age. This condition causes unnecessary and detrimental psychological distress, social isolation, and expense; the annual direct costs for caring for persons with urinary incontinence in the United States have been estimated at $16.2 billion (both men and women, 1995 US dollars).2 Surprisingly, most of this money is spent on management measures, such as diapers and pads. Current research initiatives at the University of Pittsburgh are intended to dramatically VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY 51 Tissue Engineering for SUI continued alter and improve the current standards of SUI patient care, with efforts directed at rebuilding the damaged urethra using tissue engineering strategies. The etiology of SUI appears to be multifactorial, involving changes that occur with advancing age, hormonal status, and pelvic floor damage resulting from vaginal childbirth. Given this multifactorial etiology, there are few effective nonsurgical and pharmacologic treatment options.3 Surgical treatment involving suburethral pubovaginal sling placement is one of the most common forms of treatment, with an overall objective SUI cure rate of over 80% (at 48 months and longer).4 This procedure aims to stabilize the urethrovesical junction and provide compression of the urethral lumen during stress maneuvers. Currently, graft options for sling therapy include autografts (rectus fascia, fascia lata, vaginal wall, etc), allografts (cadaveric tissues, including dura mater, dermis, fascia lata), xenografts (porcine small intestinal submucosa, porcine dermis), and synthetic materials (polytetrafluoroethylene [Gore-Tex®], graft. This additional procedure lengthens the operative time, prolongs hospitalization (secondary to postoperative pain), and increases overall costs.5 Despite the associated morbidity of the second incision, autologous fascia is advantageous with regard to the lack of vaginal erosion, a complication reported with synthetic graft materials. Synthetic materials avoid the morbidity associated with autologous harvesting procedures and clearly offer a higher tensile strength and greater persistence compared with autologous or allograft materials. However, in addition to erosion, they are associated with a higher occurrence of other complications, including infection and urethral fistula formation.6 Allograft sling materials have advantages similar to those of synthetic materials, without the higher risks of erosion and infection. In addition, allograft materials have been found to be more cost-effective than autologous fascia.7 However, there are reports of early failure of cadaveric sling materials due to fraying, resulting in recurrent SUI.8,9 Given such factors, the ideal sling material would be Allograft sling materials have advantages similar to those of synthetic materials, without the higher risks of erosion and infection. polypropylene [Marlex®], silicone elastomers [Silastic®], polyglactic acid [Vicryl®], polyester [Mersiline®]) (Table 1). Surgeon preference for graft material varies widely, and each material is associated with its own inherent advantages and disadvantages. Autografts present fewer health risks to the patient. However, this material may be inherently weak in SUI patients, and a second incision is required to procure the tissue 52 VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY devoid of such complications, readily available, durable, and lacking in antigenicity, while performing its intended function. In response to such concerns, research is under way to engineer sling grafts meeting both the physiologic and physical demands placed on them. One such proposed graft would be composed of a functional muscle/scaffolding that, when implanted, would be conducive to host tissue incorporation and continued Table 1 Currently Available Sling Materials Autografts • Rectus fascia • Fascia lata • Vaginal wall Allografts (cadaveric tissues) • Dura mater • Dermis • Fascia lata Xenografts • Porcine small intestinal submucosa • Porcine dermis Synthetics • Polytetrafluoroethylene • Polypropylene • Silicone elastomers • Polyglactic acid • Polyester remodeling while maintaining necessary strength requirements. Thus, the choice of the starting scaffolding material is critical to the success of such an approach. The Scaffold Material In an attempt to guide new tissue ingrowth and structural organization, biomaterials are frequently used as scaffolds for urologic tissue engineering.10,11 Although there are many biomaterials available, the acellular tissue matrices in particular have been proven to support cellular ingrowth and regeneration of genitourinary tissues, including the urethra and bladder tissues.12,13 Porcine small intestinal submucosa (SIS) is one such acellular biomaterial (Figure 1). SIS is derived from the submucosal layer (submucosa, muscularis mucosa, and stratum compactum) of pig small intestine that has been mechanically separated from the adjoining intestinal layers. SIS consists primarily of acellular collagen, with the original 3-dimensional structure of the natural intestinal collagen remaining intact. This Tissue Engineering for SUI 3-dimensional extracellular matrix (ECM) contains active cytokines, binding proteins, and matrix receptors that regulate the extracellular milieu to affect cellular migration, infiltration, proliferation, and differentiation. A more detailed compositional analysis indicates that the solid components of hydrated SIS (89.0% ± 1% H2O) consist of approximately 74% ± 10% protein, of which approximately 90% is collagen, as is expected of a tissue-derived ECM scaffold. The glycosaminoglycan content of lyophilized SIS is approximately 2%.14 Compositional analysis has also revealed the presence of several important scaffold glycoproteins, including laminin, entactin, and fibronectin, as well as the proteoglycans decorin and heparan sulfate proteoglycan.15 These proteins have important roles in tissue remodeling as mediators of growth factor activity. Growth factors have also been identified in SIS, including basic fibroblast growth factor (bFGF),16 acidic fibroblast growth factor (aFGF; unpublished data), vascular endothelial growth factor (VEGF),17 and transforming growth factor (TGF)-ß in both active and latent forms.18 Figure 1. A hydrated sheet of porcine small intestinal submucosa. cytokines and their regulatory substrate components that are naturally present in the ECM. • The cytokines present in SIS have been shown to attract host cells and promote angiogenesis. • Cells grown in vitro on SIS proliferate and differentiate, mimicking their in vivo responses. • SIS is strong and easily sutured and can be easily manufactured to match the physical properties, in terms of strength, porosity, and compliance, of the tissue being replaced. • SIS is biocompatible and does not produce an immunologic rejection response. Inert implants that eventually degrade leave a niche for invading pathogens to colonize. Experience with SIS implants in multiple organ systems in various animals and in vitro has demonstrated many desirable scaffold characteristics of this biomaterial: • Implanted SIS ECM promotes ingrowth of cells that remodel the ECM into functional host tissues. • SIS is recognized and remodeled by host cells because of its 3-dimensional architecture and its bioinformation content, including active Such characteristics make SIS an ideal candidate scaffolding base for the development of a functional and biologically compatible sling. SIS/Native Tissue Remodeling An implant that performs a function the day it is implanted, and continues to perform the intended function even while undergoing complete tissue infiltration, possesses distinct advantages over other more “inert” implants. When an implant is placed into a host it can undergo encapsulation, rapid resorption and dissolution, or incorporation and cellular infiltration such that the implant and the host tissues coalesce. Inert implants that eventually degrade leave a niche for invading pathogens to colonize. Implants that encapsulate offer a safe haven to such pathogens by providing an immune-privileged area that is not readily accessible to the host’s immune system. However, an implant that permits remodeling and becomes rapidly infiltrated by host cells, tissues, and blood vessels becomes completely accessible to the host, allowing defense of the implant area against opportunistic invaders. A wide variety of surgical applications in different animal species have shown that SIS implants incorporate, remodel, and eventually become entirely replaced by host tissues.19-21 In all cases, a mild inflammatory response is followed by rapid host cell infiltration, including recruitment of circulating progenitor cells from the blood,20 new blood vessel formation, and deposition of new ECM. The implant actively supports connective and epithelial tissue ingrowth and differentiation, as well as deposition, VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY 53 Tissue Engineering for SUI continued Figure 2. The processes of tissue degradation and reconstruction balance to form new tissue in and around a small intestinal submucosa (SIS) implant. The implant design incorporates these concepts into a functional device. organization, and maturation of ECM components that characterize site-specific tissue remodeling. This phenomenon has been called “smart tissue remodeling”22 and, importantly, the balance between implant degradation and host incorporation results in a dynamic implant strength response (Figure 2). An implant that has sufficient mechanical strength for tissue support yet also acts as a guide for rapid and specific tissue incorporation represents the best of both worlds with respect to both short- and long-term outcomes. Strength Characteristics Impact Clinical Outcome The physical strength of an implant, both at the time of implantation and over the life of the implant, is a critical characteristic from a clinical standpoint, particularly when the implant is intended to perform a mechanical function. Although implants may display their highest degree of strength on the day that they are implanted and subsequently weaken thereafter (such as with plastics and metals), this degradation profile is certainly not applica- 54 VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY ble to all materials. It is possible for an implant (such as SIS) to benefit from host tissue responses in such a way that the resulting implant/tissue strengthens with time; a biologic example of this strengthening effect is seen in patellar tendon autografts.23 Degradation rates that are too rapid or reconstruction rates that are too slow can result in tran- sient minimum strengths that are below a critical threshold for a given application. Therefore, both initial and long-term strength aspects of the implant must be considered depending on the particular application; furthermore, if the host response characteristics to an implant are known, the implant design can be optimized to accommodate such responses. Although strength-over-time postimplantation studies are not readily conducted in humans, this topic has been specifically addressed with regard to SIS in a canine model of body wall repair. Badylak and colleagues21,24 surgically created large abdominal wall defects and repaired them with a multilaminate, 8-layer SIS bioscaffold (processed in the same manner as the commercially available Stratasis® tension-free [TF]). The bioscaffold and the entire implant site were explanted and morphologically and mechanically examined for biaxial load-bearing capacity at times ranging from 7 days to 2 years. Figure 3. An 8-layer small intestinal submucosa (SIS) hernia repair device implanted in the canine body wall shows a dynamic strength characteristic. Implant strength diminishes approximately 40% in the first 10 days, then increases rapidly. The rate of strength increase begins to plateau after 100 days, but total wall strength is many times greater than that of normal intact canine body wall. Data from Badylak S et al. J Surg Res. 2001;99:282-287.24 Tissue Engineering for SUI Figure 3 summarizes data from this strength study, with a reference line marking the strength of the normal, unaltered canine body wall. As clearly seen in the graph, the strength of the scaffold/repair site changes with time and is characterized by a minimum strength near the 10-day mark. The strength of the repair then begins to increase over the next few months and plateaus toward an overall strength significantly greater than the normal host body wall. SIS therefore capitalizes on the host tissue response, being completely degraded and replaced with host tissue by 3 months postimplantation in this instance,21 and results in a stronger remodeled tissue at the implant site. Separate in vitro studies have also shown this material to be suitable for high load–bearing applications.25 SIS/Suburethral Grafting An SIS-based surgical implant material suitable for providing tissue support and supporting tissue incorporation in many urologic and urogynecologic procedures is currently available and is designed with a strength appropriate for providing support throughout the remodeling process (Stratasis®; Cook Urological Inc, Spencer, Ind). This material has thus far been used clinically for providing tissue support in the urologic, gynecologic, and gastroenterologic anatomy, including pubourethral support to treat SUI, repair of urethral and vaginal prolapse, bladder support, pelvic floor reconstruction, sacrocolposuspension, enterocutaneous fistula repair, and paraesophageal repair. Although longer-term clinical data relevant to the treatment of SUI are currently being collected, reported evidence thus far shows this material to be successful in providing sufficient strength and tissue support, with continence rates equal to those achieved with autologous fascia (up to 26 months follow-up).26 Of importance, SIS-based grafts are not associated with the same morbidity as autologous tissue harvesting, the risks of transmissible diseases that accompany allogeneic grafts, or the risks of infection and erosion encountered when using synthetic materials.4 Stratasis® TF, an SIS-based material specifically designed for the urethral sling procedure, incorporates several design elements that allow it to be placed without the need for anchoring sutures or bone attachment (Figure 4). At the University of Pittsburgh Medical Center, our experience with the Stratasis® sling (used in a manner similar to the tensionfree vaginal tape [TVT] slings) includes more than 20 patients with a follow-up of longer than 12 months. Although the current sample size and follow-up duration are insufficient to draw specific conclusions, results have been positive and there have been no cases of prolonged urinary retention or erosion. Furthermore, the material is easy to handle, and the placement procedure is no more difficult or timeconsuming than the traditional TVT sling procedure. Nonetheless, it is generally accepted that longer-term 5-year outcome studies are required to determine the true efficacy of a new sling material and technique. SIS/Muscle Cell Constructs Prepared In Vitro The feasibility of using SIS scaffolds to create biologic constructs in an in vitro environment that are capable of subsequent contractile function was recently explored.27 Muscle- Figure 4. Small intestinal submucosa suburethral sling placement: (A) front view and (B) side view. A B VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY 55 Tissue Engineering for SUI continued derived stem cells (MDSCs) harvested from mouse hind limb muscle were seeded onto single-layer SIS sheets (creating MDSC/SIS constructs); cultured for 1, 4, or 8 weeks; and subsequently examined histologically, as well as pharmacologically, for evaluation of isometric contractile properties. Histologic staining revealed that MDSCs could migrate into and distribute throughout the SIS, as well as form differentiated myotube structures. In addition, spontaneous contractile activity (SCA) developed in MDSC/SIS constructs by 4 and 8 weeks in culture (5/6 and 8/8 specimens, respectively) but not in 1week cultures (0/11), consistent with the time period required for maturation of the muscle structures. In contrast, SIS control groups devoid of seeded cells did not display such activity. Pharmacologic investigation of this SCA by the MDSC/SIS constructs revealed that, in most of the 4-week and in all of the 8-week preparations, both the frequency and amplitude of this SCA was decreased or completely blocked by succinylcholine in a concentration- dependent manner. This activity, however, was not affected by carbachol, potassium or, surprisingly, electrical field stimulation. Further investigation revealed SCA elimination through exposure to calciumfree solutions as well as distilled water. Thus, the contractile activity of these constructs was shown to be both calcium-dependent and modulated by nicotinic receptors. Such studies provide a glimpse of the next generation of sling devices and supply proof of concept toward the realization of an engineered in vitro biologic construct composed of specialized acellular scaffolds with desirable pre-seeded cellular populations (Figure 5). Such constructs may be useful in a number of reconstructive lower urinary tract applications, including the creation of a pubovaginal sling for the treatment of SUI that provides both structural and functional support of the deficient sphincter. Figure 5. Human urinary bladder smooth muscle cells grown for 7 days on the luminal surface of small intestinal submucosa and viewed by fluorescence microscopy: cells are dual-labeled with Oregon Green–conjugated phalloidin (cytoplasmic stain, green) and Hoechst 33342 (nuclear stain, blue). 3. 4. 5. 6. References 1. 2. Retzky SS, Rogers RM Jr. Urinary incontinence in women. Clin Symp. 1995;47:2-32. Resnick NM, Griffiths DJ. Expanding treatment options for stress urinary incontinence in women. JAMA. 2003;290:395-397. 7. Andersson KE. Drug therapy for urinary incontinence. Baillieres Best Pract Res Clin Obstet Gynaecol. 2000;14:291-313. Leach GE, Dmochowski RR, Appell RA, et al, for the American Urological Association. Female Stress Urinary Incontinence Clinical Guidelines Panel summary report on surgical management of female stress urinary incontinence. J Urol. 1997;158:875-880. Wright EJ, Iselin CE, Carr LK, Webster GD. Pubovaginal sling using cadaveric allograft fascia for the treatment of intrinsic sphincter deficiency. J Urol. 1998;160:759-762. Bidmead J, Cardozo L. Genuine stress incontinence: colpocystourethropexy versus sling procedures. Curr Opin Obstet Gynecol. 2000; 12:421-426. Labasky RF. Maybe we can do better for more—new insights on surgical candidates and techniques for incontinence surgery. J Urol. 1998;160(3 pt 1):763. Main Points • Although use of autografts for pubovaginal slings presents fewer health risks, this material may be inherently weak in patients with stress urinary incontinence, and a second incision is required to procure the tissue graft. Synthetic materials offer a higher tensile strength but have been associated with erosion, infection, and urethral fistula formation. Allograft materials present lower risks of erosion and infection and have been found to be more cost-effective than autologous fascia; however, there are reports of early failure of these materials and recurrences of incontinence. • Porcine small intestinal submucosa (SIS) exhibits several characteristics that are advantageous for sling placement: its extracellular matrix promotes ingrowth of cells that remodel it into functional host tissues; its cytokines attract host cells and promote angiogenesis; it is strong, easily sutured, and can be easily manufactured to match the physical properties of the tissue being replaced; and it is biocompatible, avoiding an immunologic rejection response. • The ability of SIS to degrade and be replaced with host tissue has been demonstrated in a canine model, in which an initial drop in implant strength was followed by an increase that was sustained, resulting in remodeled tissue stronger than the original. • Lower urinary tract tissue engineering holds promise for urologic applications other than sling procedures: one recent study showed that specialized acellular scaffolds seeded with muscle-derived stem cells were capable of spontaneous contractile activity; such technology could be applied to actual sphincter reconstruction. 56 VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY Tissue Engineering for SUI 8. 9. 10. 11. 12. 13. 14. 15. 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Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence 16. 17. 18. 19. 20. 21. 22. microscopy. J Biomater Sci Polym Ed. 2001; 12:1267-1279. Hodde JP, Hiles MC. Bioactive FGF-2 in sterilized extracellular matrix. Wounds. 2001;13:195-201. Hodde JP, Record RD, Liang HA, Badylak SF. Vascular endothelial growth factor in porcinederived extracellular matrix. Endothelium. 2001;8:11-24. McDevitt CA, Wildey GM, Cutrone RM. Transforming growth factor-beta1 in a sterilized tissue derived from the pig small intestine submucosa. J Biomed Mater Res. 2003;67A:637-640. Kropp BP, Sawyer BD, Shannon HE, et al. Characterization of small intestinal submucosa regenerated canine detrusor: assessment of reinnervation, in vitro compliance and contractility. J Urol. 1996;156:599-607. Badylak SF, Park K, Peppas N, et al. Marrowderived cells populate scaffolds composed of xenogeneic extracellular matrix. Exp Hematol. 2001;29:1310-1318. Badylak S, Kokini K, Tullius B, et al. Morphologic study of small intestinal submucosa as a body wall repair device. J Surg Res. 2002;103:190-202. Badylak SF. Small intestinal submucosa (SIS): a 23. 24. 25. 26. 27. biomaterial conducive to smart tissue remodeling. In: Bell E, ed. Tissue Engineering: Current Perspectives. Cambridge, Mass: Burkhauser Publishers; 1993:179-189. Park MJ, Lee MC, Seong SC. A comparative study of the healing of tendon autograft and tendon-bone autograft using patellar tendon in rabbits. Int Orthop. 2001;25:35-39. Badylak S, Kokini K, Tullius B, Whitson B. Strength over time of a resorbable bioscaffold for body wall repair in a dog model. J Surg Res. 2001;99:282-287. Gloeckner DC, Sacks MS, Billiar KL, Bachrach N. Mechanical evaluation and design of a multilayered collagenous repair biomaterial. J Biomed Mater Res. 2000;52:365-373. Colvert JR 3rd, Kropp BP, Cheng EY, et al. The use of small intestinal submucosa as an off-theshelf urethral sling material for pediatric urinary incontinence. J Urol. 2002;168:1872-1876. Lu SH, Cannon TW, Chermanski C, et al. Muscle-derived stem cells seeded into acellular scaffolds develop calcium-dependent contractile activity that is modulated by nicotinic receptors. Urology. 2003;61:1285-1291. VOL. 6 NO. 2 2004 REVIEWS IN UROLOGY 57