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

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.

The effect of Resveratrol and Octreotide on peritoneal adhesions in a rat model.

INTRODUCTION: The aim of this study was to investigate the efficacy of resveratrol and octreotide, agents that are used to prevent intra-abdominal adhesions in experimental models, in preventing intraperitoneal adhesions when used alone or in combination. MATERIALS AND METHODS: The study employed 28 young female Wistar albino rats weighing 250-300 grams. An experimental adhesion model was created in each rat using serosal abrasion and peritoneal excision. They were divided into four groups, each comprising seven rats: Group 1, adhesion induction only; Group 2, resveratrol administration only; Group 3, octreotide administration only; and Group 4, administration of resveratrol and octreotide combination. The rats were monitored under appropriate conditions for 14 days and then underwent laparotomy. Macroscopic intensity and extensiveness of adhesions and microscopic changes in the granulation tissue (cellular intensity, reticular and collagen fibers, capillaries, elastic and smooth muscle fibers, fibrosis) were evaluated and graded. Kruskal-Wallis and Mann-Whitney U-test were used in statistical analysis and the level of statistical significance was established as p <0.05. RESULTS: There was no significant difference between the groups in terms of the intensity and extensiveness of macroscopic adhesions (p=0.377 and p=0.319). There was a statistically significant difference between the microscopic scores of the groups according to Zühlke's classification (p=0.026). The Bonferroni correction used to test for the differences revealed that the rats in Group 1 achieved significantly higher scores than the rats in Group 3 (p=0.016). CONCLUSION: Octreotide showed higher efficiency compared to the control group in microscopic classification; however, the two agents were not superior to each other or their combination was not superior in preventing intra-abdominal adhesions.

Fiber density of collagen grafts impacts rabbit urethral regeneration.

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.

Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment.

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.

IGF-1-containing multi-layered collagen-fibrin hybrid scaffolds for bladder tissue engineering.

UNLABELLED: Clinical success of bladder reconstructive procedures could be promoted by the availability of functional biomaterials. In this study, we have developed a multi-layered scaffold consisting of a bioactive fibrin layer laminated between two collagen sheets all having undergone plastic compression. With this construct we performed bladder augmentation in a nude rat model after partial bladder excision and evaluated the morphological and functional behavior of the implant. The fibrin was functionalized with a recombinant human insulin-like growth factor-1 (IGF-1) variant that covalently binds fibrin during polymerization and has a matrix metalloproteinase-cleavage insert to enable cell-mediated release. The purified IGF-1 variant showed similar bioactivity in vitro compared to commercially available wild type (wt) IGF-1, inducing receptor phosphorylation and induction of human smooth muscle cell proliferation. In vivo, the multi-layered bioactive collagen-fibrin scaffolds loaded with the IGF-1 variant triggered dose-dependent functional host smooth muscle cell invasion and bundle formation with re-urothelialization 4weeks after surgery in a rat model. STATEMENT OF SIGNIFICANCE: The design of new bio-functional scaffolds that can be employed for bladder reconstructive procedures is a growing focus in the field of tissue engineering. In this study, a fibrin binding form of human insulin-like growth factor-1 (IGF-1) was produced and used to functionalize a multi-layered collagen-fibrin scaffold consisting of bioactive fibrin layer, sandwiched between two collagen gels. An effective dosage of our IGF-1 variant was successfully determined via a nude rat bladder model, which may play a critical role in estimating its therapeutic dosage in clinical trials. Thus, this new bioactive scaffold may offer an advanced approach to accelerate bladder regeneration.

Histochemical and immunohistochemical characteristics of elastofibromas.

Elastofibromas are slow-growing and rare soft-tissue tumors. The etiology and pathogenetic mechanisms are still controversial and there are only a few studies in the literature investigating the histochemical, immunohistochemical, and genetic features to determine the pathogenesis. We investigated the cellular composition of lesions with a diagnosis of elastofibroma in 17 patients by using histochemical and immunohistochemical methods. There were 17 cases with a mean age of 53.5 years. Mean lesion diameter was 6.6 cm. The immunohistochemical method showed vimentin and factor XIIIa positivity in all cases. Four cases had focal myoglobin positivity in the spindle-shaped cells between the collagen fibers. Spindle cells were positive for CD34 in 8 cases. Smooth muscle actin, desmin, type 4 collagen and laminin were negative in all cases. The elastic nature of the abnormal fibers was shown histoch with Verhoeff elastin staining and aldehyde fuchsin staining in all cases. Our results have shown that the concurrent positivity of factor XIIIa and CD34 in the cells forming the lesion might show that the lesionoriginates from primitive dermal mesenchymal cells. In addition, the myoglobin positivity found in some cases indicates the possibility of a myofibroblastic origin of elastofibromas.

[The efficacy and toxicity of gemcitabine and cisplatin chemotherapy in advanced/metastatic bladder urothelial carcinoma]. / La eficacia y toxicidad de la quimioterapia con gemcitabina y cisplatino en el carcinoma urotelial de vejiga avanzado/metastásico.

OBJECTIVE: Many new agents have been introduced as an alternative to standard MVAC therapy with improved efficacy and lower toxicity profile in advanced bladder carcinoma. The aim of this study is to evaluate the response rate and toxic side effects of gemcitabine-cisplatin (GC) in patients with advanced/metastatic bladder carcinoma. METHODS: Between January 2001 and April 2006, 58 patients with histologically confirmed advanced/metastatic transitional cell carcinoma (TCC) were enrolled in the study. All patients received 1,000 mg/m(2) gemcitabine administered via intravenous infusion of 30-60 minutes on days 1, 8 and 15, and 70 mg/m(2) cisplatin as an infusion of 60-min on day 2. All toxicities were graded using the WHO scale and the National Cancer Institute scale. RESULTS: The average number of cycles was 4.1. Neutropenia and thrombocytopenia were clinically significant treatment-related side-effects. Hematologic toxicity included mainly grade 3-4 neutropenia in 56%, grade 3-4 thrombocytopenia in 59%, and grade 3- 4 anemia in 33% of patients. There was only one death from neutropenic sepsis. Complete response and partial response were obtained in 13 (22.4%) and 17 (29.3%) of patients, respectively, 17 (29.3%) of patients were found to have stable disease, and progression was observed in 11 patients (18.9%). Median survival for the whole group was 14.7 months (2-67). CONCLUSIONS: GC therapy is an effective regimen owing to its high tumor response and long survival with a low incidence of toxicity in advanced or metastatic patients.

Porous Agarose-Based Semi-IPN Hydrogels: Characterization and Cell Affinity Studies.

Hydrogels are frequently considered for medical applications due to the ease of preparation in different forms and high water content that makes them comparable to natural tissues. However, these general properties are not sufficient to make any hydrogel suitable for cell attachment and growth which are necessary for their use in tissue regeneration. Besides, the high water content makes the hydrogels mechanically weak. The formation of semi-interpenetrating networks (semi-IPNs) can be used in attempts to enhance physical, mechanical and thermal properties. In this study, semi-IPNs of agarose were prepared with chitosan and alginate, two polyelectrolytes that are positively and negatively charged under physiological conditions, respectively. Zeta potential was used to confirm the formation of charged hydrogels. All hydrogels had ultimate compression strengths in the range of 91-210 Pa where the value for pure agarose was about 103 Pa. Chitosan increased the compressive strength about two folds whereas the alginate had opposite effects. The amount of strongly bound water present in the hydrogels were estimated from TGA and DSC analysis and the highest value was found for alginate-agarose hydrogels as about 15%. The attachment and the migration of L929 fibroblasts were monitored in vitro using the MTS assay and confocal microscopy. The highest cell proliferation and penetration were observed for positively charged chitosan-agarose semi-IPN hydrogels.

Infliximab in experimental alkali burns of the oesophagus in the rat.

BACKGROUND: We aimed to investigate the efficacy of infliximab, a chimeric TNF-alpha antibody, in the prevention of fibrosis in an experimental alkaline burn of the oesophagus in the rat. METHODS: Thirty-two Wistar albino rats divided into four experimental groups. Caustic oesophageal burn was induced by applying 37.5% NaOH to the distal oesophagus. Infliximab was given at a dose of 5 mg/kg via the intraperitoneal route. Group A (sham) animals were uninjured, group B had untreated oesophageal burns, group C had oesophageal burns treated with a single dose of infliximab on the first day, and Group D had oesophageal burns treated with infliximab on the first and 14th days. Efficacy of the treatment was assessed on the 28th-day by measuring stenosis index of the oesophagus and histopathological damage score, and biochemically by determining tissue hydroxyproline content. RESULTS: There was no significant difference between the Group B and the infliximab treated Groups C and D in means of tissue hydroxyproline content and histopathological damage scores. Stenosis index was not significantly different between the Group B, Group C, and Group D. CONCLUSION: Anti-TNF-alpha treatment with infliximab does not ameliorate the degree of fibrosis in alkali burns of the oesophagus in the rat. Further evaluation of inflammatory and immunological events leading to stricture in alkaline oesophageal burns may provide new perspectives for the treatment of alkaline oesophageal burns.

beta-catenin gene mutation in invasive ductal breast cancer.

PURPOSE: Aberrant accumulation of beta-catenin plays an important role in a variety of human neoplasms. In this study we analyzed the somatic mutations of the beta-catenin gene and the immunohistochemical localization of beta-catenin and cyclin D1 in invasive ductal breast cancer. MATERIALS AND METHODS: We investigated 65 human invasive ductal breast cancer samples for somatic mutations in the exons 3, 4, 5 and 6 of beta-catenin gene (N-terminal region) by the combined use of polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) and sequencing. Sample tissues were also analyzed using beta-catenin and cyclin D1 immunocytochemistry staining. RESULTS: No beta-catenin mutation was detected in any of the tumor samples. Accumulation of aberrant beta-catenin protein in cellular compartments in the same breast cancer samples was confirmed with a related experiment by immunocytochemical methods. CONCLUSION: Our results suggest that genetic defects in beta-catenin is not common in invasive ductal breast cancers, whereas mutations in other components of the Wnt signaling pathway should be considered.

Hydatid cysts with unusual localizations: diagnostic and treatment dilemmas for surgeons.

Although the liver and lung are by far the most common localizations for the larval cysts of Echinococcus granulosus in humans, the cysts may develop in other sites and there cause signs and symptoms that may be easily confused with those of other illnesses. In a retrospective study in Turkey, six male and 11 female cases of cystic echinococcosis, each with at least one cyst in an unusual site, were investigated. The patients, who had a mean age of 41.6 years, had cysts in the pancreas, intra-abdominal cavity, kidney, spleen, ovary, breast, mediastinum, chest wall, muscle and/or subcutaneous tissue. In terms of Gharbi's classification, 15 (75%) of the 20 cysts in these patients were type I and five (25%) were type II. Fourteen of the cases each had single cysts, two had multiple cysts, and one had an unknown number of cysts. All but one of the cases (who had a pancreatic cyst) were treated by total cystectomy. In areas where cystic echinococcosis is endemic, any patient presenting with a cystic mass, in any tissue or organ, should be considered a potential case of the disease and be carefully investigated by radio-imaging and/or ultrasonography.

The relation of mutant p53 accumulation in transitional cell carcinoma of bladder with pathological stage, grade, recurrence and survival.

In this study prognostic significance and clinical value of mutant p53 gene in bladder transitional cell tumors is investigated. In our clinic, between 1997-2000, transurethral resection was performed in 48 cases, 3 females (6%) and 45 males (94%) with the diagnosis of primary bladder tumor, age ranges between 30-81 years old (average 58.9 +/- 9.9). The patients whose pathology results were transitional cell carcinoma were gathered into two groups as p53 positive and p53 negative by immunohistochemical study. These cases who were followed 1-36 months were compared to each other for pathologic state, tumor grade, recurrence and survival. It's found out that mutant p53 accumulation is related to high grade and pathologic stage tumors. But it's concluded that p53 positivity doesn't effect recurrence and survival rates.