BACKGROUND: A variety of tissue engineering techniques are currently under development or investigation for bladder augmentation, but so far no approach is clearly superior. The aim of this study was to compare the suitability for cystoplasty augmentation in rats of in vivo implanted acellular bladder matrices (BAM) previously seeded with hair follicle stem cells and that of matrices implanted without the cells. MATERIALS AND METHODS: The rat hair follicle stem cell line was positive for CD34, p63, and Ki-67. 1 x 10(6) cells from 34 to 40 passages seeded onto nine BAM scaffolds were cultured for one week. Nine other scaffolds were left unseeded. Scaffolds were grafted into a surgically created defect within the anterior bladder wall: nine rats with acellular matrices and nine with cell-seeded BAM. Rats observed for six months were killed in monthly intervals. We performed gross examination, X-ray cystography, and hematoxylin-eosin, cytokine (CK)-7, CK-20, myoglobin, and desmin staining of the excised bladders. RESULTS: Minimal adhesions were observed and urinary leakage was noted in one case. Two animals died in the acellular group. Rats developed stone disease in bladders reconstructed with acellular BAM. Bladder capacity was similar, but the shape was regular and characteristically oval only in bladders grafted with cell-seeded BAM. Muscle layers in the apical parts of the reconstructed bladder walls were extremely thin in the cases of acellular grafts and thicker in bladders reconstructed with cell-seeded grafts. Muscle layer regeneration was better in the cell-seeded group. Urothelium regenerated in all animals. CONCLUSIONS: We have shown that hair follicle stem cells may be used for rat bladder wall regeneration.

BACKGROUND: In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts. METHODS: Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed. RESULTS: Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45). CONCLUSIONS: Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.

Several platinum(IV) complexes are showing considerable promise in initial trials, producing reactive intermediates that then interact with DNA. AIM: To perform in vitro study of two new platinum(IV) complexes cytotoxic effect on B16 mouse melanoma cells. METHODS: PtCl(4)(dbtp)(2) and PtCl(2)(6mp)(2) complexes were prepared. PtCl(4)(dbtp)(2) was created as modification of PtCl(4)(dmtp) test previously. Apoptosis and necrosis were examined using flow cytometry, upon Annexin V/PI staining. RESULTS: LC(10), LC(50) and LC(90) parameters established for PtCl(4)(dbtp)(2) were as following: 2.6, 17.0, 58.0 mumol/L. However LC(10) and LC(50) established for PtCl(2)(6mp)(2) were 1.2 and 14.0 mumol/l respectively. The both complexes induced apoptosis. PtCl(2)(6mp)(2) induced cell cycle arrest in G0/G1, while PtCl(4)(dbtp)(2)- in S-phase. Conclusions: PtCl(4)(dbtp)(2) appeared to be more cytotoxic against B16 cells than PtCl(2)(6mp)(2). Apoptosis was the main mechanism of cell loss in cultures incubated with both tested complexes.

Abdominal hernia repair is one of the most common surgical procedures. Current data indicate that the best treatment results are achieved with use of synthetic material to reinforce weakened abdominal wall. Prosthetic materials utilized for hernia repair induce adhesions with underlying viscera. They should be therefore separated from them by a layer of peritoneum otherwise adhesions may cause to serious complications such as bowel-skin fistulas. The aim of our work was to determine if implantation of our collagen membrane into abdominal wall defect induce adhesions in rat model of ventral hernia. The collagen film was obtained by acetic acid extraction of rat tail tendons and than casting the soluble fraction onto polyethylene shits. Abdominal wall defect was created in 10 Wistar male rats. Collagen membranes were implanted into the defect using interrupted polypropylene stitches. After 3 months of observation all animals were sacrificed. No adhesions between path structure and bowel developed. In one often rats (10%) adhesion between fixating stitch and omentum was observed. Complete mesothelium lining and vascular ingrowth were microscopically observed within implanted structure. Promising result requires further confirmation in a larger series of animals.

PURPOSE OF INVESTIGATION: The aim of the study was to determine the activity of cathepsin D (CTSD) and alpha-1-antitrypsin (AAT) in the blood serum of women with cervical carcinoma treated with different modes of therapy. METHODS: The study was conducted on 68 women suffering from carcinoma of the uterine cervix, that were irradiated intracavitarily by a Selectron LDR brachytherapy unit. Additionally, all patients were treated with different therapy methods according to clinical stage. RESULTS: In women with cervical cancer, CTSD activity was higher while AAT activity was lower both before and after brachytherapy sessions as compared to controls. Six months after the end of therapy, the activity of CTSD and AAT reverted back to the values characteristic for healthy women. CONCLUSION: The estimation of cathepsin D and alpha-1-antitrypsin activity during the course of cervical cancer management may be useful in early detection of potential recurrence and/or widespread metastasis formation.

Pancreatic islet transplantation is a treatment method for type I diabetes. Its outcome is influenced by numerous factors, islet quantity and function being important ones of them. THE AIM OF THIS STUDY: was to estimate the influence of pancreas preparation method on the outcome of islet isolation in rat. MATERIAL AND METHODS: 6 pancreata harvested from Lewis rats were used in this research. Pancreatic duct was cannulated and pancreas was injected with 1 mg/ml collagenase P solution (Sigma) and then excised. After cutting into smaller fragments, it was digested in collagenase P solution for 15-20 min. Enzyme activity was then stopped by adding dilution medium. Heterogenous cell suspension was centrifuged in density gradient (Gradisol) to isolate islets. Pancreatic islets were collected and islet equivalent was calculated. Islet purity degree was estimated as islet cells to all cells, including exocrine, ratio. Islet viability was estimated using propidium iodide and fluorescein diacetate staining. Photographic documentation was made. RESULTS: Proper islet morphology, highest number and viability was obtained when pancreas was excised properly (isolation 3 and 4). CONCLUSION: Pancreas preparation method is one of which influences on islet isolation outcome.

It was shown that ointments containing silver compounds delay wound healing and increase the risk of hypertrophic scarring in burned patients. The aim of the study was to establish the influence of AgNO3 on cell viability and apoptosis of keratinocytes and fibroblasts in vitro. Foreskin was used to establish primary human keratinocyte and fibroblasts cell cultures. Keratynocytes grew in DMEM/Ham's-12 at 3:1 ratio, supplemented with 10% FBS, EGF, insulin, transferin, triiodothyronine and hydrocortisone. Fibroblasts were cultured in DMEM with addition of 10% FBS. The influence of AgNO3 on keratinocyte and fibroblast cultures was evaluated by fluorescence microscopy and flow cytometry. Double staining with Annexin V-FITC and propidium iodide was performed. The AgNO3 at lower concentration (3 and 15 x 10(-4) M/dm3) than used for patient's treatment (31 x 10(-4) M/dm3) revealed to be toxic and trigger apoptosis in human keratinocytes and fibroblasts. The ointments containing nitrates should be used with caution especially in conditions where the epithelial layer is destroyed. The nitrate can negatively affect wound healing.