Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

Karolinska Institutet

Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. In addition to being important in antimicrobial defense, it induces chemotaxis, stimulates angiogenesis and promotes tissue repair. We previously showed that human breast cancer cells express high amounts of hCAP18, and hypothesised that hCAP18/LL-37 may be involved in tumour progression.

Human antimicrobial protein hCAP18/LL-37 promotes a metastatic phenotype in breast cancer.

Surgical intervention is sometimes needed to create a conduit from the abdominal wall to the bladder for self-catheterization. We developed a method for tissue engineering a conduit for bladder emptying without in vitro cell culturing as a one-step procedure. In a porcine animal model bladder, wall tissue was excised and the mucosa was minced to small particles. The particles were attached to a tube in a 1 : 3 expansion rate with fibrin glue and transplanted back by attaching the tube to the bladder and through the abdominal wall. Sham served as controls. After 4-5 weeks, conduits were assessed in respect to macroscopic and microscopic appearance in 6 pigs. Two pigs underwent radiology before termination. Gross examination revealed a patent conduit with an opening to the bladder. Histology and immunostaining showed a multilayered transitional uroepithelium in all cases. Up to 89% of the luminal surface area was neoepithelialized but with a loose attachment to the submucosa. No epithelium was found in control animals. CT imaging revealed a patent channel that could be used for filling and emptying the bladder. Animals that experienced surgical complications did not form conduits. Minced autologous bladder mucosa can be transplanted around a tubular mold to create a conduit to the urinary bladder without in vitro culturing.

Transplantation of autologous minced bladder mucosa for a one-step reconstruction of a tissue engineered bladder conduit.

Cultured human urothelial cells can be used in tissue engineering for reconstruction of urothelial defects. For safety reasons, a fine characterization of the cells is required before use in reconstructive surgery. For these reasons, we aimed to characterize the effect of in vitro propagation of urothelial cells on gene expression and proliferative capacity. Gene expression of urothelial cells in passage two and eight was captured by using a microarray chip covering the whole human genome. To find relationships in biological functions and pathways, differentially regulated genes were subjected to pathway analysis using the WEB-based Gene Set Analysis Toolkit (WebGestalt). Proliferative capacity was tested with population doubling time, efficiency in colony formation assays, and immunocytochemistry. In addition, senescence markers were evaluated. Bioinformatics analysis revealed gene expression profile differences. Downregulated genes at passage eight clustered in biological pathways of cell cycle and DNA repair processes; upregulated genes had no obvious association to any specific biological function or pathway according to WebGestalt analysis, but individual genes with extracellular matrix, apoptosis, and cell morphology. Data were supported by reverse transcription-polymerase chain reaction (RT-PCR) and in vitro growth experiments. Passage two urothelial cells had higher efficiency in colony formation and lower population doubling time. An increase in senescence markers was detected at passage eight. We conclude that pretransplantation quality controls are important and, for reconstructive purposes, cells should be transplanted back to the patient as soon as possible to procure good proliferative capacity also after transplantation.

A study on proliferation and gene expression in normal human urothelial cells in culture.

In order to develop autologous tissue engineering of the whole wall in the urinary excretory system, we studied the regenerative capacity of the muscular bladder wall. Smooth muscle cell expansion on minced detrusor muscle  and  with or without urothelial tissue was studied. Porcine minced detrusor muscle and urothelium were cultured  under standard culture conditions for evaluation of the explant technique and in collagen for tissue sectioning and histology. Autografts of minced detrusor muscle with or without minced urothelium were expanded on 3D cylinder moulds by grafting into the subcutaneous fat of the pig abdominal wall. Moulds without autografts were used as controls. Tissue harvesting, mincing, and transplantation were performed as a one-step procedure. Cells from minced detrusor muscle specimens migrated and expanded  on culture plastic and in collagen.  studies with minced detrusor autografts demonstrated expansion and regeneration in all specimens. Minced urothelium autografts showed multilayered transitional urothelium when transplanted alone but not in cotransplantation with detrusor muscle; thus, minced bladder mucosa was not favored by cografting with minced detrusor. No regeneration of smooth muscle or epithelium was seen in controls.

Expansion of Submucosal Bladder Wall Tissue  and .

Quality control studies addressing gene expression changes and genetic stability are of vital importance in regenerative medicine. In order to rule out that in vitro expansion gives rise to gene expression changes that could favour oncogenic events, this study applied a total human gene expression chip (Affymetrix®) and bioinformatics analysis using the Ingenuity web-based application in combination with an analysis of chromosomal copy number variations using array comparative genomic hybridization. Urothelial cells presented a general repression of genes required for cell cycle progression and upregulation of growth-inhibitory genes, as well as a decrease in deoxyribose nucleic acid replication after long-term culture. Molecules were identified with a potential to regulate human urothelial cell senescence, such as the micro-ribonucleic acid Let-7. Human urothelial cells did not acquire copy number variations after long-term culture and the cells had a normal expression of oncogenes and tumor suppressor genes. Altogether, both gene expression studies and array comparative genomic hybridization indicated a good quality of in vitro propagated cells. For tissue engineering purposes, these analyses could be used for quality control assessments before transplantation back to the patient. Copyright © 2016 John Wiley & Sons, Ltd.

Study on genetic stability in human urothelial cells in vitro.

The success of regenerative medicine relies in part on the quality of the cells implanted. Cell cultures from cells isolated from bladder washes have been successfully established, but molecular changes and cell characteristics have not been explored in detail. In this work, we analysed the role of telomere shortening in relation to the regenerative potential and senescence of cells isolated from bladder washes and expanded in culture. We also analysed whether bladder washes would be a potential source for attaining stem cells or promoting stem cell proliferation by using two different substrates to support their growth: a feeder layer of growth-arrested murine fibroblasts J2 3T3 cells and a xeno-free human recombinant laminin-coated surface. We found no association between telomere shortening and senescence in urothelial cells in vitro. Urothelial cells had a stable telomere length and expressed mesenchymal stem cells markers but failed to differentiate into bone or adipocytes. Feeder layer showed an advantage to laminin-coated surfaces in respect to proliferative capacity with the expense of risking that feeder layer cells could persist in later passages. This emphasizes the importance of using carefully controlled culture conditions and molecular quality controls before autotransplantation in future clinical settings. In conclusion, urothelial cells isolated by bladder washes show regenerative potential that need further understanding. Senescence in vitro might be due to cellular stress, and if so, further improvements in culture conditions may lead to longer cell life and higher proliferative capacity.

Urothelial cell senescence is not linked with telomere shortening.

 Tissue engineering is a potential source of urethral substitutes to treat severe urethral defects. Our aim was to create tissue-engineered urethras by harvesting autologous cells obtained by bladder washes and then using these cells to create a neourethra in a chronic large urethral defect in a rabbit model.  A large urethral defect was first created in male New Zealand rabbits by resecting an elliptic defect (70 mm) in the ventral penile urethra and then letting it settle down as a chronic defect for 5-6 weeks. Urothelial cells were harvested noninvasively by washing the bladder with saline and isolating urothelial cells. Neourethras were created by seeding urothelial cells on a commercially available decellularized intestinal submucosa matrix (Biodesign® Cook-Biotech®). Twenty-two rabbits were divided into three groups. Group-A ( = 2) is a control group (urethral defect unrepaired). Group-B ( = 10) and group-C ( = 10) underwent on-lay urethroplasty, with unseeded matrix (group-B) and urothelial cell-seeded matrix (group-C). Macroscopic appearance, radiology, and histology were assessed.  The chronic large urethral defect model was successfully created. Stratified urothelial cultures attached to the matrix were obtained. All group-A rabbits kept the urethral defect size unchanged (70 ± 2.5 mm). All group-B rabbits presented urethroplasty dehiscence, with a median defect of 61 mm (range 34-70). In group-C, five presented complete correction and five almost total correction with fistula, with a median defect of 0.3 mm (range 0-12.5), demonstrating a significant better result ( = 7.85 × 10). Urethrography showed more fistulas in group-B (10/10, versus 5/10 in group-C) ( = 0.04). No strictures were found in any of the groups. Group-B histology identified the absence of ventral urethra in unrepaired areas, with squamous cell metaplasia in the edges toward the defect. In group-C repaired areas, ventral multilayer urothelium was identified with cells staining for urothelial cell marker cytokeratin-7.  The importance of this study is that we used a chronic large urethral defect animal model and clearly found that cell-seeded transplants were superior to nonseeded. In addition, bladder washing was a feasible method for harvesting viable autologous cells in a noninvasive way. There is a place for considering tissue-engineered transplants in the surgical armamentarium for treating complex urethral defects and hypospadias cases.

Creation of Tissue-Engineered Urethras for Large Urethral Defect Repair in a Rabbit Experimental Model.

Urinary bladder wound healing relies on multiple biological events that are finely tuned in a spatial-temporal manner. MicroRNAs are small non-coding RNA molecules with regulatory functions. We hypothesized that microRNAs are important molecules in the coordination of normal urinary bladder wound healing. We aimed at identifying microRNAs expressed during bladder wound healing using Affymetrix global array for microRNA profiling of the rodent urinary bladder during healing of a surgically created wound. Results were validated in the rat bladders by real-time PCR (RT-PCR) using three of the differentially expressed (DE) microRNAs. The model was thereafter validated in human cells, by measuring the expression of eight of the DE microRNAs upon in vitro wound-healing assays in primary urothelial cells. Our results indicated that 508 (40%) of all rodent microRNAs were expressed in the urinary bladder during wound healing. Thirteen of these microRNAs (1%) were DE (false discovery rate (FDR) < 0.05, P < 0.05, |logfold|> 0.25) in wounded compared to non-wounded bladders. Bioinformatic analyses helped us to identify target molecules for the DE microRNAs, and biological pathways involved in tissue repair. All data are made available in an open-access database for other researchers to explore.

A database on differentially expressed microRNAs during rodent bladder healing.

In severe malformations with a lack of native tissues, treatment options are limited. We aimed at expanding tissue in vivo using the body as a bioreactor and developing a sustainable single-staged procedure for autologous tissue reconstruction in malformation surgery. Autologous micro-epithelium from skin was integrated with plastically compressed collagen and a degradable knitted fabric mesh. Sixty-three scaffolds were implanted in nine rats for histological and mechanical analyses, up to 4 weeks after transplantation. Tissue integration, cell expansion, proliferation, inflammation, strength, and elasticity were evaluated over time in vivo and validated in vitro in a bladder wound healing model. After 5 days in vivo, we observed keratinocyte proliferation on top of the transplant, remodeling of the collagen, and neovascularization within the transplant. At 4 weeks, all transplants were fully integrated with the surrounding tissue. Tensile strength and elasticity were retained during the whole study period. In the in vitro models, a multilayered epithelium covered the defect after 4 weeks. Autologous micro-epithelial transplants allowed for cell expansion and reorganization in vivo without conventional pre-operative in vitro cell propagation. The method was easy to perform and did not require handling outside the operating theater.

Exploring the Concept of In Vivo Guided Tissue Engineering by a Single-Stage Surgical Procedure in a Rodent Model.

Urogenital reconstructive surgery can be impeded by lack of tissue. Further developments within the discipline of tissue engineering may be part of a solution to improve clinical outcomes. In this study, we aimed to design an accessible and easily assembled tubular graft with autologous tissue, which could be constructed and implanted as a single-staged surgical procedure within the premises of an ordinary operating room. The ultimate goals would be to optimize current treatment-options for long-term urinary diversion. Therefore, we evaluated the optimal composition of a collagen-based scaffold with urothelial micrografts in vitro, and followingly implanted the construct in vivo as a bladder conduit. The scaffold was evaluated in relation to cell regeneration, permeability, and biomechanical properties. After establishing an optimized scaffold in vitro, consisting of high-density collagen with submerged autologous micrografts and reinforced with a mesh and stent, the construct was successfully implanted in an in vivo minipig model. The construct assemblance and surgical implantation proved feasible within the timeframe of a routine surgical intervention, and the animal quickly recovered postoperatively. Three weeks post-implantation, the conduit demonstrated good host-integration with a multilayered luminal urothelium. Our findings have encouraged us to support its use in more extensive preclinical large-animal studies.

Clara Ibel Chamorro

Laboratory of Tissue Engineering,
Department of Women's and Children's Health,
Laboratory of Tissue Engineering, Department of Women's and Children's Health

Karolinska Institutet

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Clara Ibel Chamorro

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Tissue Engineering,Department of Women's and Children's Health,Laboratory of Tissue Engineering, Department of Women's and Children's Health

Karolinska Institutet

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Laboratory of Tissue Engineering,
Department of Women's and Children's Health,
Laboratory of Tissue Engineering, Department of Women's and Children's Health