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

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, 2 cm 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 9 months 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 2 cm 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. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model

Peter Frey, Jeffrey Alan Hubbell, Lionel Ambroise Micol

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies. (C) 2012 Elsevier Ltd. All rights reserved.

Elsevier2012

Fiber density of collagen grafts impacts rabbit urethral regeneration

Peter Frey, Jeffrey Alan Hubbell, Hans Mattias Larsson, Kalitha Pinnagoda, Elif Vardar, Ganesh Vythilingam

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.

2018

Is There a Need for Smooth Muscle Cell Transplantation in Urethral Reconstruction?

Peter Frey

Background: Hypospadias and urethral strictures are conditions requiring additional tissue for reconstruction. Due to a limited source of tissue, autologous skin and oral mucosa are frequently used. However, long-term follow-up studies demonstrated significant complications and diminished quality of life. Recently, a variety of tubular biodegradable biomaterials have been used. Cell seeding seems to be important to improve the host acceptance and neovascularization. Objective: To compare in vivo performance of smooth muscle cell (SMC)-seeded and unseeded tubular collagen-based scaffolds in a rabbit urethral reconstruction model. Materials and Methods: Sixteen New Zealand rabbits underwent an open-bladder biopsy for SMC harvesting. The SMCs were cultured for 3 weeks and labeled with ethynyldeoxyuridine (EdU). A 1-cm-length tubular collagen-based 0.5wt% scaffold was seeded and cultured with SMCs and implantation in a rabbit model. Eight rabbits received SMC-seeded scaffolds for a 1-cm-length circumferential urethral repair, situated 1.5cm from the meatus. After 1 and 3 months, four rabbits underwent a urethrography and were sacrificed. The penises underwent hematoxylin and eosin, immunohistochemistry, and EdU fluorescence staining. In the control group eight rabbits received acellular scaffolds. Results: The SMC-seeded group presented one stricture at 1 month and one fistula at 3 months. Three strictures were present in the unseeded group at 1 month and one at 3 months. In the seeded group, more SMC expression and neovascularization was observed, and less mononuclear and giant cells could be found. All scaffolds showed luminal urothelial cell revetment. The detection of EdU-labeled SMCs revealed SMC transplantation survival. Conclusion: SMC-seeded tubular collagen scaffolds improved urethral regeneration in this rabbit model. Such constructs may be valuable for repair of severe urethral diseases.

Mary Ann Liebert, Inc2014

Is There a Need for Smooth Muscle Cell Transplantation in Urethral Reconstruction?

Peter Frey

Mary Ann Liebert, Inc2014