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

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

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Studies of the role of the cathelicidin peptide LL-37 in epithelial biology

The biology of epithelial immunity as well as of tissue repair is complex and highly regulated. A variety of molecules, cell types, and biological processes such as, cell differentiation, proliferation and migration, programmed cell death and antimicrobial mechanisms contribute to maintaining a balance between tissue damage and tissue repair. Different sources of evidence indicated that the cathelicidin hCAP18/LL-37 might, not only act as an antibacterial molecule, but as a key regulator of the above mentioned processes. We therefore tested this concept through in vivo and in vitro studies aimed at determining whether and how hCAP18/LL-37 could be such a multifunctional molecule. The goal of this thesis was to investigate the role of LL-37 in epithelial cell biology by addressing the following questions: How is the expression of the human cathelicidin gene (CAMP) controlled in primary keratinocytes? Does LL-37 contribute to tissue repair through either, regulation of cell proliferation, differentiation and/or programmed cell death? Which genes become expressed in the presence of LL-37 and what potential biological processes do they control? And does LL-37 contribute to defects in cell proliferation, differentiation and/or migration during pathological states such as cancer? We found that the active form of Vitamin D (VD) and its metabolites induced the expression of CAMP. As the biological effects of VD are mediated by the vitamin D receptor (VDR), which, once activated binds to response elements in the promoter region of target genes (VDRE), we tested the CAMP promoter for the presence of VDR binding sites. We identified one active VDRE binding site at about ~500 bp from the transcription start site. In vivo stimulation of human skin with the vitamin D analog calcipotriol resulted in high expression of hCAP18 at the mRNA and protein levels, compared to control skin samples from the same individuals (Paper I). Tissue homeostasis is maintained by, among others, the selective removal of cells through mechanisms such as apoptosis or programmed cell death. Because cell proliferation and differentiation are central to skin biology, we tested the role of LL-37 in the apoptosis of human keratinocytes. We found that LL-37 prevented camptothecin-induced apoptosis, which was associated with decreased caspase-3 activity and increased expression of PGE2 and IAP2, through a mechanism dependent on COX2 activity (Paper II). By using a microarray approach we found that LL-37 affected the gene expression profile of human keratinocytes with a signifficant effect on STC2. We investigated the mechanism of LL-37-induced STC2 upregulation and found evidence (paper III, manuscript) suggesting that unfolded protein response in keratinocytes might be triggered by expossure to LL-37. We measured the expression of the hCAP18/LL-37 in a panel of 104 breast cancer tumors and compared it to the levels found in control samples, as well as analyzed its relationship to clinical data. We also studied, in vitro, the effect of LL-37 on breast cancer cell migration and in anchorage independent colony formation. In addition, we explored the molecular mechanisms responsible for LL-37 effects on breast cancer cells. To evaluate the relevance of our findings in vivo, a xenograft model using severely compromised immunodeficient (SCID) mice was designed to test the effect of the transgenic expression of the hCAP18/LL-37 in tumor formation. The results from these experiments indicate that 1) hCAP18/LL-37 is functionally connected with ErbB2; 2) LL-37 alters breast cancer cell phenotype in vitro 3) LL-37 stimulates the migration of breast cancer cells and 4) LL-37 stimulates metastasis formation in SCDI mice in vivo (paper IV). Increasing evidence shows that besides its immune function, LL-37 has tissue-repair-like effects, promotes cell proliferation, migration and angiogenesis. Furthermore LL-37 has been implicated in the pathogenesis of inflammatory skin disease such as psoriasis. Based on our findings, we propose that LL-37 is a key regulator of epithelial homeostasis by influencing, tissue defense, tissue repair and maintenance through control of programmed cell death, in association with vitamin D and likely acting as an alarmin by activating processes such as the unfolded protein response. Thus, these effects become relevant for the study of pathological states such as chronic inflammation and cancer

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

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

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

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