Eva-Maria Engelhardt

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

Compressed collagen gel: a novel scaffold for human bladder cells

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. Copyright

IGF-1-containing multi-layered collagen-fibrin hybrid scaffolds for bladder tissue engineering

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.

In vivo study of an injectable poly(acrylonitrile)-based hydrogel paste as a bulking agent for the treatment of urinary incontinence.

Urinary incontinence can be treated by endoscopic injection of bulking agents, however, no optimal therapeutic effect has been achieved upon this treatment yet. In the present study, the development of a injectable poly(acrylonitrile) hydrogel paste is described, and its efficacy and histological behavior, once injected into the submucosal space of the minipig bladder, are evaluated. A device was developed to mix poly(acrylonitrile) hydrogel powder with glycerin, used as carrier, prior to injection into the submucosal space of the bladder. Several paste deposits, depending on the size of the bladder, were injected per animal. The implants were harvested at days 7, 14, 21, 28, 84 and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated. However, at later time points the implants were split up and surrounded by granulomatous tissue, which was gradually replaced by histiocytes and adipocytes. Transitory focal urothelial metaplasia was observed only at day 7 and moderate foreign body reaction was detected predominantly between the second and fifth week. This study demonstrated the feasibility to develop an injectable paste of poly(acrylonitrile) hydrogel thought to provide the expected bulking effect, necessary for the treatment of urinary incontinence.

Engineered acellular collagen scaffold for endogenous cell guidance, a novel approach in urethral regeneration

UNLABELLED The treatment of congenital malformations or injuries of the urethra using existing autologous tissues can be associated with post-operative complications. Using rat-tail collagen, we have engineered an acellular high-density collagen tube. These tubes were made of 2 layers and they could sustain greater burst pressures than the monolayered tubes. Although it remains a weak material this 2 layered tube could be sutured to the native urethra. In 20 male New Zealand white rabbits, 2cm long grafts were sutured in place after subtotal excision of the urethra. This long-term study was performed in Lausanne (Switzerland) and in Kuala Lumpur (Malaysia). No catheter was placed post-operatively. All rabbits survived the surgical implantation. The animals were evaluated at 1, 3, 6, and 9months by contrast voiding cysto-urethrography, histological examination and immunohistochemistry. Spontaneous re-population of urothelial and smooth muscle cells on all grafts was demonstrated. Cellular organization increased with time, however, 20% of both fistula and stenosis could be observed post-operatively. This off-the shelf scaffold with a promising urethral regeneration has a potential for clinical application. STATEMENT OF SIGNIFICANCE In this study we have tissue engineered a novel cell free tubular collagen based scaffold and used it as a urethral graft in a rabbit model. The novelty of our technique is that the tube can be sutured. Testing showed better burst pressures and the grafts could then be successfully implanted after a urethral excision. This long term study demonstrated excellent biocompatibility of the 2cm graft and gradual regeneration with time, challenging the current literature. Finally, the main impact is that we describe an off-the-shelf and cost-effective product with comparable surgical outcome to the cellular grafts.

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

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.

Application of Polyvinylidene Fluoride (PVDF) as a Biomaterial in Medical Textiles

ABSTRACT Textile implants so called scaffolds are used for the reinforcement of tissue on the one hand and cell guidance on the other hand with the goal to support regeneration of new tissue. Different factors strongly influence cell ingrowth of the scaffold namely the fibre material the fibre structure and the textile structure Depending on the final application of a textile scaffold warp knitting weavingbraiding and non-woven technologies can be used to manufacture the textile structure. Thermal treatment can be applied to adjust the porosity geometry and elasticity of the produced textiles. At the Institut fur Texiltechnik at RWTH Aachen University (RWTH-ITΑ) polyvinylidene fluoride (PVDF) is used as a non-degradable and biocompatible polymer. PVDF shows very good biocompatibility and even bioneutrality. For the application in medical products, the PVDF is spun by a melt spinning process. In addition to multi- and monofilament also bicomponent fibres can be produced. Multifilaments are subject of currently carried out studies at the ITA. These multifilaments are used for medical applications like vascular grafts, ligament and artificial cornea. The artificial cornea is presented in detail. In another study, scaffolds made out of PVDF fibres with varying diameter and cross section are produced. The influence of the miscellaneous scaffolds on cell adhesion is then investigated.

Fiber density of collagen grafts impacts rabbit urethral regeneration.

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.

Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment

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 μm 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.

GFP-Expressing Bladder Fibroblasts for Applications in Tissue Engineering

The availability of green fluorescent protein (GFP)-expressing primary cells is of great interest for the monitoring of cell growth on and within three-dimensional (3D) scaffolds for tissue engineering applications. We used piggyBac (PB)-transposon-mediated nucleofection for gene delivery to generate primary bladder fibroblasts that stably express the GFP gene. Alternatively, cells were transduced with a lentivirus vector carrying the GFP gene. Homogenous GFP-positive cell populations were obtained by cell sorting (lentivirus transduction) or by puromycin selection (PB-mediated gene delivery). Both methods resulted in stable GFP-positive cell pools. However, instability of GFP expression was observed in some pools originating from nucleofection. The successful generation of GFP-expressing human bladder fibroblasts allowed us to monitor cell growth on poly(lactic acid-ɛ-caprolactone) scaffolds using GFP-specific fluorescence as a surrogate marker for cell number.