RESUMEN
Stress urinary incontinence (SUI) is a life changing condition, affecting 20 million women worldwide. In this study, we developed a bioactive, injectable bulking agent that consists of Permacol™ (Medtronic, Switzerland) and recombinant insulin like growth factor-1 conjugated fibrin micro-beads (fib_rIGF-1) for its bulk stability and capacity to induce muscle regeneration. Therefore, Permacol™ formulations were injected in the submucosal space of rabbit bladders. The ability of a bulking material to form a stable and muscle-inducing bulk represents for us a promising therapeutic approach to achieve a long-lasting treatment for SUI. The fib_rIGF-1 showed no adverse effect on human smooth muscle cell metabolic activity and viability in vitro based on AlamarBlue assays and Live/Dead staining. Three months after injection of fib_rIGF-1 together with Permacol™ into the rabbit bladder wall, we observed a smooth muscle tissue like formation within the injected materials. Positive staining for alpha smooth muscle actin, calponin, and caldesmon demonstrated a contractile phenotype of the newly formed smooth muscle tissue. Moreover, the fib_rIGF-1 treated group also improved the neovascularization at the injection site, confirmed by CD31 positive staining compared to bulks made of PermacolTM only. The results of this study encourage us to further develop this injectable, bioactive bulking material towards a future therapeutic approach for a minimal invasive and long-lasting treatment of SUI.
Asunto(s)
Materiales Biocompatibles/uso terapéutico , Incontinencia Urinaria de Esfuerzo/terapia , Animales , Materiales Biocompatibles/química , Femenino , Fibrina/química , Humanos , Inmunohistoquímica , Ratones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Conejos , Incontinencia Urinaria de Esfuerzo/metabolismo , Sistema Urinario/citología , Sistema Urinario/metabolismoRESUMEN
There is a need for efficient and "off-the-shelf" grafts in urethral reconstructive surgery. Currently available surgical techniques require harvesting of grafts from autologous sites, with increased risk of surgical complications and added patient discomfort. Therefore, a cost-effective and cell-free graft with adequate regenerative potential has a great chance to be translated into clinical practice. Tubular cell-free collagen grafts were prepared by varying the collagen density and fiber distribution, thereby creating a polarized low fiber density collagen graft (LD-graft). A uniform, high fiber density collagen graft (HD-graft) was engineered as a control. These two grafts were implanted to bridge a 2 cm long iatrogenic urethral defect in a rabbit model. Histology revealed that rabbits implanted with the LD-graft had a better smooth muscle regeneration compared to the HD-graft. The overall functional outcome assessed by contrast voiding cystourethrography showed patency of the urethra in 90% for the LD-graft and in 66.6% for the HD-graft. Functional regeneration of the rabbit implanted with the LD-graft could further be demonstrated by successful mating, resulting in healthy offspring. In conclusion, cell-free low-density polarized collagen grafts show better urethral regeneration than high-density collagen grafts.
Asunto(s)
Colágeno/metabolismo , Ingeniería de Tejidos/métodos , Uretra/patología , Animales , Fibras de la Dieta , Matriz Extracelular , Masculino , Modelos Animales , Músculo Liso , Conejos , Procedimientos de Cirugía Plástica , Regeneración , Trasplantes/metabolismo , Trasplantes/cirugía , Uretra/trasplanteRESUMEN
Endoscopic injection of bulking agents has been widely used to treat urinary incontinence, often due to urethral sphincter complex insufficiency. The aim of the study was to develop a novel injectable bioactive collagen-fibrin bulking agent restoring long-term continence by functional muscle tissue regeneration. Fibrin micro-beads were engineered using a droplet microfluidic system. They had an average diameter of 140⯵m and recombinant fibrin-binding insulin-like growth factor-1 (α2PI1-8-MMP-IGF-1) was covalently conjugated to the beads. A plasmin fibrin degradation assay showed that 72.5% of the initial amount of α2PI1-8-MMP-IGF-1 loaded into the micro-beads was retained within the fibrin micro-beads. In vitro, the growth factor modified fibrin micro-beads enhanced cell attachment and the migration of human urinary tract smooth muscle cells, however, no change of the cellular metabolic activity was seen. These bioactive micro-beads were mixed with genipin-crosslinked homogenized collagen, acting as a carrier. The collagen concentration, the degree of crosslinking, and the mechanical behavior of this bioactive collagen-fibrin injectable were comparable to reference samples. This novel injectable showed no burst release of the growth factor, had a positive effect on cell behavior and may therefore induce smooth muscle regeneration in vivo, necessary for the functional treatment of stress and other urinary incontinences. STATEMENT OF SIGNIFICANCE: Urinary incontinence is involuntary urine leakage, resulting from a deficient function of the sphincter muscle complex. Yet there is no functional cure for this devastating condition using current treatment options. Applied physical and surgical therapies have limited success. In this study, a novel bioactive injectable bulking agent, triggering new muscle regeneration at the injection site, has been evaluated. This injectable consists of cross-linked collagen and fibrin micro-beads, functionalized with bound insulin-like growth factor-1 (α2PI1-8-MMP-IGF-1). These bioactive fibrin micro-beads induced human smooth muscle cell migration in vitro. Thus, this injectable bulking agent is apt to be a good candidate for regeneration of urethral sphincter muscle, ensuring a long-lasting treatment for urinary incontinence.
Asunto(s)
Colágeno/química , Reactivos de Enlaces Cruzados/química , Fibrina/uso terapéutico , Inyecciones , Microfluídica/métodos , Microesferas , Incontinencia Urinaria/tratamiento farmacológico , Animales , Movimiento Celular , Supervivencia Celular , Módulo de Elasticidad , Fibrina/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Tamaño de la Partícula , Ratas , Reología , Incontinencia Urinaria/patología , ViscosidadRESUMEN
UNLABELLED: Clinical success of bladder reconstructive procedures could be promoted by the availability of functional biomaterials. In this study, we have developed a multi-layered scaffold consisting of a bioactive fibrin layer laminated between two collagen sheets all having undergone plastic compression. With this construct we performed bladder augmentation in a nude rat model after partial bladder excision and evaluated the morphological and functional behavior of the implant. The fibrin was functionalized with a recombinant human insulin-like growth factor-1 (IGF-1) variant that covalently binds fibrin during polymerization and has a matrix metalloproteinase-cleavage insert to enable cell-mediated release. The purified IGF-1 variant showed similar bioactivity in vitro compared to commercially available wild type (wt) IGF-1, inducing receptor phosphorylation and induction of human smooth muscle cell proliferation. In vivo, the multi-layered bioactive collagen-fibrin scaffolds loaded with the IGF-1 variant triggered dose-dependent functional host smooth muscle cell invasion and bundle formation with re-urothelialization 4weeks after surgery in a rat model. STATEMENT OF SIGNIFICANCE: The design of new bio-functional scaffolds that can be employed for bladder reconstructive procedures is a growing focus in the field of tissue engineering. In this study, a fibrin binding form of human insulin-like growth factor-1 (IGF-1) was produced and used to functionalize a multi-layered collagen-fibrin scaffold consisting of bioactive fibrin layer, sandwiched between two collagen gels. An effective dosage of our IGF-1 variant was successfully determined via a nude rat bladder model, which may play a critical role in estimating its therapeutic dosage in clinical trials. Thus, this new bioactive scaffold may offer an advanced approach to accelerate bladder regeneration.
Asunto(s)
Colágeno/farmacología , Fibrina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Vejiga Urinaria/fisiología , Animales , Materiales Biocompatibles/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Ratas Desnudas , Vejiga Urinaria/cirugíaRESUMEN
Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.
Asunto(s)
Colágeno/farmacología , Fuerza Compresiva/efectos de los fármacos , Andamios del Tejido/química , Vejiga Urinaria/citología , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Secciones por Congelación , Geles , Humanos , Inmunohistoquímica , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/ultraestructura , Plásticos/farmacología , Ratas , Propiedades de Superficie/efectos de los fármacos , Urotelio/citología , Urotelio/ultraestructuraRESUMEN
Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.