Modification of the Human Amniotic Membrane Using Different Cross-Linking Agents as a Promising Tool for Regenerative Medicine.

Human amniotic membranes (hAMs) obtained during cesarean sections have proven to be clinically useful as an interesting biomaterial in a wide range of tissue engineering applications such as ocular surface reconstruction, burn treatments, chronic wounds, or bedsore ulcers. It presents antimicrobial properties, promotes epithelization, reduces inflammation and angiogenesis, contains growth factors, and constitutes the reservoir of stem cells. However, variability in hAM stiffness and its fast degradation offers an explanation for the poor clinical applications and reproducibility. In addition, the preparatory method of hAM for clinical use can affect its mechanical properties, and these differences can influence its application. As a directly applied biomaterial, the hAM should be available in a ready-to-use manner in clinical settings. In the present study, we performed an analysis to improve the mechanical properties of hAM by the addition of various reagents used as protein cross-linkers: EDC/NHS, PEG-dialdehyde, PEG-NHS, dialdehyde starch, and squaric acid. The effect of hAM modification using different cross-linking agents was determined via infrared spectroscopy, thermal analyses, mechanical properties analyses, enzymatic degradation, and cytotoxicity tests. The use of PEG-dialdehyde, PEG-NHS, dialdehyde starch, and squaric acid increases the mechanical strength and elongation at the breaking point of hAM, while the addition of EDC/NHS results in material stiffening and shrinkage. Also, the thermal stability and degradation resistance were evaluated, demonstrating higher values after cross-linking. Overall, these results suggest that modification of human amniotic membrane by various reagents used as protein cross-linkers may make it easier to use hAM in clinical applications, and the presented study is a step forward in the standardization of the hAM preparation method.

Urinary bladder augmentation with acellular biologic scaffold-A preclinical study in a large animal model.

Current strategies in urinary bladder augmentation include use of gastrointestinal segments, however, the technique is associated with inevitable complications. An acellular biologic scaffold seems to be a promising option for urinary bladder augmentation. The aim of this study was to evaluate the utility of bladder acellular matrix (BAM) for reconstruction of clinically significant large urinary bladder wall defects in a long-term porcine model. Urinary bladders were harvested from 10 pig donors. Biological scaffolds were prepared by chemically removing all cellular components from urinary bladder tissue. A total of 10 female pigs underwent hemicystectomy and subsequent bladder reconstruction with BAM. The follow-up study was 6 months. Reconstructed bladders were subjected to radiological, macroscopic, histological, immunohistochemical, and molecular evaluations. Six out of ten animals survived the 6-month follow-up period. Four pigs died during observation due to mechanical failure of the scaffold, anastomotic dehiscence between the scaffold and native bladder tissue, or occluded catheter. Tissue engineered bladder function was normal without any signs of postvoid residual urine in the bladder or upper urinary tracts. Macroscopically, graft shrinkage was observed. Urothelium completely covered the luminal surface of the graft. Smooth muscle regeneration was observed mainly in the peripheral graft region and gradually decreased toward the center of the graft. Expression of urothelial, smooth muscle, blood vessel, and nerve markers were lower in the reconstructed bladder wall compared to the native bladder. BAM seems to be a promising biomaterial for reconstruction of large urinary bladder wall defects. Further research on cell-seeded BAM to enhance urinary bladder regeneration is required.

New Amniotic Membrane Based Biocomposite for Future Application in Reconstructive Urology.

OBJECTIVE: Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. MATERIAL AND METHODS: Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. RESULTS: Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson's trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young's elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. CONCLUSIONS: Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.

Radical prostatectomy specimens - a voice against focal therapy.

INTRODUCTION: The main treatment methods of prostate carcinoma are surgery and radiation therapy, both having serious side effects. Because of these side effects, the idea of organ preserving therapy emerged. Rationale to perform focal therapy is to preserve the prostate gland, along with potency and continence, offering good cancer control with appropriate treatment. The idea of gland sparing therapy is quite controversial. Presently, EAU Guidelines propose focal therapy as experimental in the treatment of prostate carcinoma. MATERIAL AND METHODS: The aim of the study was to asses how many patients could be qualified for focal therapy, according to post prostatectomy pathological findings. 720 patients suspected of prostate cancer were biopsied. In 324 patients, prostate carcinoma was revealed, of which 81 were subjected to radical prostatectomy. Pre and post-operative pathological results were analyzed, according to possible qualification for focal treatment. RESULTS: According to the clinical evaluation of all the patients referred to the treatment, 25% could be assumed to have unifocal disease and could be qualified to the focal treatment. Post-operative evaluation revealed pT2b cancer in 5%, pT2c disease in 65%, and pT3a-pT4a disease in 20% of these patients. Cancer was unilateral (pT2a-b) in only 15% of cases, and was suitable for focal treatment (small disease not extending to whole lobe- pT2a disease) in only 10%. CONCLUSIONS: It seems that with the use of current methods, proper T-staging of the disease and amount of neoplasmatic tissue inside the gland can not be reached with great certainty. In our opinion, focal therapy should not be used in patients with ≤pT2b and high risk disease. For them, radical treatment (surgery or radiation therapy) should be recommended. For the rest of the patients, with low risk disease, keeping in mind the large scale of possible overtreatment, active surveillance is a valid treatment option. Focal therapy can be an interesting therapeutic proposition for a small group of patients with pT2a cancer, but it is not possible to select them with big certainty with current methods of imaging medicine.

The time from diagnosis of bladder cancer to radical cystectomy in Polish urological centres - results of CysTiming Poland study.

INTRODUCTION: The aim of the study was to assess the waiting time, from establishing the indications for radical cystectomy to surgery, in patients with urothelial carcinoma of the bladder at different Polish urological centres and to determine its influencing factors. MATERIAL AND METHODS: Retrospective analysis of data was performed on all consecutive radical cystectomies, performed in 2008-2012, at 10 Polish urological centres. The waiting time of patients from establishing the indications for radical cystectomy to surgery, as well as factors potentially influencing this time, were assessed. University (3), provincial (3) and regional (4) hospitals were defined as the 3(rd), 2(nd) and 1(st) level referral hospitals, respectively. RESULTS: A total of 575 patients qualified for radical cystectomy due to muscle invasive urothelial carcinoma of the bladder (MIBC, 68% of cases) or failure of previous treatment of non-muscle invasive urothelial carcinoma of the bladder (NMIBC, 32%) were included in the analysis. The average time after the establishment of indications to surgery was 73.4 days, with a median of 56 days. In the case of 121 patients (22.1%), the waiting time exceeded 90 days. Significant differences in waiting time were found when the hospital referral levels were taken into consideration. In the 3(rd) level referral hospitals the median time for cystectomy was 61.5 days (p = 0.035), in the 2(nd) level referral hospitals - 45 days (p = 0.000) and, in the 1(st) level referral hospitals - 58 days (p = 0.051). CONCLUSIONS: The waiting time from establishing the indications for radical cystectomy to surgery for most cases in Poland does not exceed 90 days.

Do mesenchymal stem cells modulate the milieu of reconstructed bladder wall?

To evaluate the mesenchymal stem cells (MSCs) influence on cytokines and matrix metalloproteinases (MMPs) expression in rat bladder wall regeneration. MSCs cultures from the bone marrow were established. Acellular matrices from the bladder submucosa were prepared. Bladders were reconstructed using cell-seeded (n = 5) and unseeded (n = 5) grafts. MSCs were injected into the bladder wall (n = 5), bladders were incised and MSCs were injected into the circulation (n = 5) or were left intact (n = 5). Animals were killed after 3 months. Bladder histology and immunohistochemical staining of IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-ß1, IFN-γ, MMP-2, and MMP-9 were done. Bladders reconstructed with cell-seeded grafts mimicked native tissue, while unseeded grafts revealed shrinkage and morphological irregularities. There were no morphological changes in bladders of other groups. Different pattern of cytokine and MMP expression was observed. Increased expression of anti-inflammatory cytokines and MMPs in bladder promotes detrusor regeneration.

Ablative therapies for small renal tumors.

Ablative therapies of renal tumors are steadily gaining popularity in clinical practice due to the many benefits they offer to patients. Moreover, ablative procedures hold promise in the field of uro-oncology for the best compromise between low invasiveness, high efficacy and advantages in terms of procedural costs. Reported outcomes with ablative therapies for small renal tumors are excellent and without significant differences for surgical procedures based on nephron-sparing surgery. Nevertheless, these methods for treatment of small renal tumors should still be confined to carefully selected patients. This review discusses the currently used ablative techniques in urology.

The relationship of cancer stem cells in urological cancers.

Numerous studies are ongoing to identify and isolate cancer stem cells from cancers of genito-urinary tracts. Better understanding of their role in prostate, urothelial and kidney cancer origin, growth and progression opens new pathways in development of more effective treatment methods. However there are still many issues before advances in this field can be introduced for clinical application. This review addresses current achievements in cancer stem cells research in uro-oncology.

Ciprofloxacin is a potential topoisomerase II inhibitor for the treatment of NSCLC.

Lung cancer is one of the most common tumors and its treatment is still inefficient. In our previous work we proved that ciprofloxacin has a different influence on five cancer cell lines. Here, we aimed to compare the biological effect of ciprofloxacin on cell lines representing different responses after treatment, thus A549 was chosen as a sensitive model, C6 and B16 as highly resistant. Three different cell lines were analyzed (A549, B16 and C6). The characterization of continuous cell growth was analyzed with the Real-Time Cell Analyzer (RTCA)-DP system. Cytoskeletal changes were demonstrated using immunofluorescence. The cell cycle was analyzed using flow cytometry. Ciprofloxacin was cytostatic only against the A549 cell line. In the case of other tested cell lines a cytostatic effect was not observed. Cytoskeletal analysis confirms the results obtained with RTCA-DP. A549 cells were inhibited in the G2/M phase suggesting a mechanism related to topoisomerase II inhibition. The biological effects of ciprofloxacin support the hypothesis that this drug can serve as an adjuvant treatment for lung cancer, due to its properties enabling topoisomerase II inhibition.

Schwann cells - a new hope in tissue engineered urinary bladder innervation. A method of cell isolation.

INTRODUCTION: There are not any effective method to induce the innervation of urinary bladder wall graft after augmentation. Neurons from urinary bladder wall and omentium can not elongate and branch in graft because of lack of neurotrophic factors. The best source of these neurotrophic factors are Schwann cells which can be transplanted into urinary bladder wall graft. To transplant Schwann cells the proper amount of cells is needed which can be only obtain during in vitro Schwann cell cultivation. We introduce the results of Schwann cell isolation and in vitro cultivation. MATERIALS AND METHODS: 33 Wistar rats, males (350 gr.) were used in this study. Animal were divited into two groups (n = 15). Cell cultures were established in both groups on 5, 6, 7, 8 nad 9 day after nerve injury. In first group the digestion time with colagenase and trypsyne was 2.5 h and in second one 3.5 h. RESULTS: A larger number of cells were isolated from the degenerated sciatic nerve. Colonies of cells that morphologically resembled Schwann cells were visible by light microscopy on the second day of in vitro cultivation. Homogeneity of the primary cultures increased in the last day of cultivation to 60%. CONCLUSIONS: Schwann cells isolation from predegenerated peripheral nerve is effective and can delivered require amount of cells for transplantation to urinary bladder graft.