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

Pinnagoda K, Larsson HM, Vythilingam G, Vardar E, Engelhardt EM, Thambidorai RC, et al.

Acta Biomater, 2016 10 01;43:208-217.
PMID: 27450527 DOI: 10.1016/j.actbio.2016.07.033

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.
Fulltext Fiber density of collagen grafts impacts rabbit urethral regeneration

Larsson HM, Vythilingam G, Pinnagoda K, Vardar E, Engelhardt EM, Sothilingam S, et al.

Sci Rep, 2018 07 03;8(1):10057.
PMID: 29968749 DOI: 10.1038/s41598-018-27621-9

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.
A bioactive injectable bulking material; a potential therapeutic approach for stress urinary incontinence

Vardar E, Vythilingam G, Pinnagoda K, Engelhardt EM, Zambelli PY, Hubbell JA, et al.

Biomaterials, 2019 06;206:41-48.
PMID: 30925287 DOI: 10.1016/j.biomaterials.2019.03.030

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.
Fulltext Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix

Zielinski MS, Vardar E, Vythilingam G, Engelhardt EM, Hubbell JA, Frey P, et al.

Commun Biol, 2019;2:69.
PMID: 30793047 DOI: 10.1038/s42003-019-0313-x

By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.
Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment

Vardar E, Larsson HM, Allazetta S, Engelhardt EM, Pinnagoda K, Vythilingam G, et al.

Acta Biomater, 2018 02;67:156-166.
PMID: 29197579 DOI: 10.1016/j.actbio.2017.11.034

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.