Surgical Implantation of Single-Staged Tissue-Engineered Urothelial Tubes in a Minipig Model.

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

A perioperative layered autologous tissue expansion graft for hollow organ repair.

Tissue engineering has not been widely adopted in clinical settings for several reasons, including technical challenges, high costs, and regulatory complexity. Here, we introduce the Perioperative Layered Autologous Tissue Expansion graft (PLATE graft), a composite biomaterial and collagen-reinforced construct with autologous epithelium on one side and smooth muscle tissue on the other. Designed to mimic the structure and function of natural hollow organs, the PLATE graft is unique in that it can be produced in a standard operating theatre and is cost-effective. In this proof-of-principle study, we test its regenerative performance in eight different organs, present biomechanical and permeability tests, and finally explore its in vivo performance in live rabbits.

Advancing autologous urothelial micrografting and composite tubular grafts for future single-staged urogenital reconstructions.

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.

Insights into cellular behavior and micromolecular communication in urothelial micrografts.

Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the proliferative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.

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

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.

A database on differentially expressed microRNAs during rodent bladder healing.

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.

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

Introduction: 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. Methods: A large urethral defect was first created in male New Zealand rabbits by resecting an elliptic defect (70 mm2) 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 (n = 2) is a control group (urethral defect unrepaired). Group-B (n = 10) and group-C (n = 10) underwent on-lay urethroplasty, with unseeded matrix (group-B) and urothelial cell-seeded matrix (group-C). Macroscopic appearance, radiology, and histology were assessed. Results: 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 mm2). All group-B rabbits presented urethroplasty dehiscence, with a median defect of 61 mm2 (range 34-70). In group-C, five presented complete correction and five almost total correction with fistula, with a median defect of 0.3 mm2 (range 0-12.5), demonstrating a significant better result (p = 7.85 × 10-5). Urethrography showed more fistulas in group-B (10/10, versus 5/10 in group-C) (p = 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. Conclusions: 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.

Molecular and histological studies of bladder wound healing in a rodent model.

The field of regenerative medicine encounters different challenges. The success of tissue-engineered implants is dependent on proper wound healing. Today, the process of normal urinary bladder wound healing is poorly characterized. We aspired to explore and elucidate the natural response to injury in an in vivo model in order to further optimize tissue regeneration in future studies. In this study, we aimed to characterize histological and molecular changes during normal healing in a rat model by performing a standardized incisional wound followed by surgical closure. We used a rodent model (n = 40) to follow the healing process in the urinary bladder for 28 days. Surgical exposure of the bladder without incision (n = 40) was performed in controls. Histological characterization and western blot analyses of proteins was carried out using specific staining and markers for inflammation, proliferation, angiogenesis, and tissue maturation. For the molecular characterization of gene expression total RNA was collected for RT2 -PCR in wound healing pathway arrays. Analysis of histology revealed distinct, but overlapping, phases of healing with a local inflammatory response (days 1-8) simultaneous with a rapid formation of granulation tissue and proliferation (days 2-8). We also identified significant changes in gene expression related to inflammation, proliferation, and extracellular matrix formation. Healing of an incisional wound in a rodent urinary bladder demonstrated that all the classical phases of wound healing: hemostasis, inflammation, proliferation followed by tissue maturation were present. Our data suggest that the bladder and the skin share similar molecular signaling during wound healing, although we noted differences in the duration of each phase compared to previous studies in rat skin. Further studies will address whether our findings can be extrapolated to the human bladder.

Urothelial cell senescence is not linked with telomere shortening.

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.

Resección completa de neurocitoma extraventricular hipervascularizado tras embolización preoperatoria / Complete resection of hypervascularised extraventricular neurocytoma after preoperative embolisation

Una mujer de 13 años de edad presenta clínica de cefalea de 15 días de evolución y solo edema de papila bilateral en la exploración. El estudio inicial de tomografía computarizada y RM mostró una gran masa multiquística frontal izquierda con calcificación rodeada de edema periférico, sangrado intralesional subagudo y múltiples importantes vasos asociados. Se interviene en otro centro, encontrando cavidad con hematoma subagudo que se evacua con múltiples vasos y venas arteriolizadas. Ante la sospecha de malformación arteriovenosa (MAV) a pesar de los hallazgos de la neuroimagen realizada previamente, se deriva a nuestro centro para seguir tratamiento. Realizamos arteriografía, angio-RM y RM con secuencias avanzadas que muestran masa intraaxial hipervascularizada que se emboliza previo a la interviene quirúrgica definitiva con resultado anatomopatológico de neurocitoma extraventricular (NEV). Los NEV son lesiones extremadamente raras que no se han descrito previamente en la literatura como lesiones hipervascularizadas que en nuestro caso requirió la realización de angiografía y embolización previa para su correcto diagnóstico y adecuado manejo

A 13-year-old female arrived at the Emergency Department with a two-week history of headache, and bilateral papilloedema on examination. The initial study with CT and MRI showed a large multicystic left frontal mass with calcification surrounded by peripheral oedema, subacute intralesional bleeding and association of multiple large vessels. She was initially operated on in another centre where a subacute haematoma was found, evacuating to multiple vessels and arteriolised veins. Despite the earlier neuroimaging findings, arteriovenous malformation (AVM) was suspected, so she was referred to our centre for further treatment. We performed angiography, MR angiography and MRI with advanced sequences, diagnosing a highly vascularised intra-axial tumour which was embolised. The patient was then definitively operated on, with the resulting finding of extraventricular neurocytoma (EVN). EVN are extremely rare lesions, not previously described in the literature as hypervascularised lesions, which in our case required prior angiography and embolisation for proper diagnosis and adequate management

Complete resection of hypervascularised extraventricular neurocytoma after preoperative embolisation. / Resección completa de neurocitoma extraventricular hipervascularizado tras embolización preoperatoria.

A 13-year-old female arrived at the Emergency Department with a two-week history of headache, and bilateral papilloedema on examination. The initial study with CT and MRI showed a large multicystic left frontal mass with calcification surrounded by peripheral oedema, subacute intralesional bleeding and association of multiple large vessels. She was initially operated on in another centre where a subacute haematoma was found, evacuating to multiple vessels and arteriolised veins. Despite the earlier neuroimaging findings, arteriovenous malformation (AVM) was suspected, so she was referred to our centre for further treatment. We performed angiography, MR angiography and MRI with advanced sequences, diagnosing a highly vascularised intra-axial tumour which was embolised. The patient was then definitively operated on, with the resulting finding of extraventricular neurocytoma (EVN). EVN are extremely rare lesions, not previously described in the literature as hypervascularised lesions, which in our case required prior angiography and embolisation for proper diagnosis and adequate management.

Study on genetic stability in human urothelial cells in vitro.

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.

Expansion of Submucosal Bladder Wall Tissue In Vitro and In Vivo.

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 in vitro and in vivo with or without urothelial tissue was studied. Porcine minced detrusor muscle and urothelium were cultured in vitro 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 in vitro on culture plastic and in collagen. In vivo 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.

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Ovarian Small Cell Carcinoma of Pulmonary Type Arising in Mature Cystic Teratomas With Metastases to the Contralateral Ovary.

A bilateral small cell ovarian carcinoma pulmonary-type (SCCOPT), arising in bilateral mature cystic teratomas (MCTs) presented as stage IIIB in a 37-year-old woman. Microscopically, tumor nests were related to the dermoid protuberance and expressed pancytokeratin, EMA, CD56, chromogranin A, NSE, synaptophysin, and SOX2. SALL4 was also focally positive. CDX2, TTF1, PAX8, CK7, CK20, and several neuroendocrine gut hormones were negative. Serum NSE was elevated. This case represents a SCCOPT arising in an MCT in the right ovary with metastasis to the left one also containing a synchronous MCT. Surface implants and lymphovascular invasion suggested metastasis from the right ovarian SCCOPT and excluded a metastatic origin from usual locations of small cell carcinoma (SCC). SCCOPT is morphologically identical to SCC elsewhere, even sharing NSE serum elevation. Although the tumor was closely related to teratomatous mature tissues, a complex immunohistochemical panel failed to provide a tissue of origin.

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

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.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients.

BACKGROUND: Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5' CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. METHODS: Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). RESULTS: Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. CONCLUSION: Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient's resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

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

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.