861 Use of bone marrow derived mesenchymal stem cells for autologous tissue engineering of urinary bladder muscularis

625

861 USE OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS FOR AUTOLOGOUS TISSUE ENGINEERING OF URINARY BLADDER MUSCULARIS

THE EFFECT ON BLADDER INTERSTITIAL

Schultheiss D.‘, Gabouev A.‘, Pilatz A.‘, Schanz J.‘, Mertsching Griensven M.V.‘, Schlote N.‘, Wefer J.‘, Stief C.‘, Jonas U.’

Acar

H.‘, Lorenr R.‘, Winkler

M.‘,

‘Harmover MedIcal School, Department of Urology, Hannover, Germany, ?Hannover Medical School, Leibniz Laboratories for Biotechnology and Artificial Organs, Hannover, Germany, ‘Hatmover Medical School, Department of Visceral and Transplantation Surgery, Hatmover, Germany, ?Hannover Medical School, Department of Trauma Surgery. Harmover, Germany & OBJECTIVES: A key question in tissue engineering is the generation of a sufficient amount of autologous primary cells for the reseeding procedure. Bone marrow derived mesenchymal stem cells (MSC) may be an alternative cell source for this. The aim of the present study was to isolate these cells and to direct them into myogenic differentiated functional cells that could be used in reconstruction of the bladder.

INTRODUCTION

& .METHODS: Porcine bone marrow was aspirated from the iliac crest and lower vertebrae. From this MSC were purified using a Percoll-gradient and amplified in culture up to two passages. They were differentiated towards myogenic cells by adding 5ngiml transforming growth factor-beta1 (TGF-betal) to the standard smooth muscle cell growth medium or by using conditioned culture medium, i.e. medium exposed to adult bladder smooth muscle cells (SMC) before use. Control groups of MSC were cultured in endothelial cell growth medium. Cell characterization was performed by immunostaining for a-&in, myosin and desmm Functionality was evaluated using photomicroscopy to visualize cell contractions after exposure of the cultures to cholinergic dmgs (10mM carbachol). Cultures of adult SMC and fibroblasts served as controls. Acellular small intestinal submucosa (SE) was reseeded using the differentiated MSC cultures and evaluated histologically. Cellular metabolic activity was measured by sequential MTS-formazan assays after one and two weeks.

MATERIAL

1” and 2”d passage cultures of MSC supplemented with TGF-beta1 and those calmred with conditioned smooth muscle cell medium showed an increased amount of immunochemical cellular markers typical of myogenic cells than MSC cultured with standard medium. Control MSC cultured in endothelial medium was lacking myogenic markers. Cholinergic drugs could induce cell contractions in up to 20% of the cells comparable to adult SMC, whereas this was not seen in fibroblast control cultures. SIS repopulated and cultured with MSC revealed myogenic cells in histology. Cell metabolic activity of these matrices was corresponding to those reseeded with the same density of adult SMC and showed a time-dependent increase of activity.

RESULTS:

The use of myogenic differentiated bone marrow derived MSC teems to be a promising alternative for functional tissue engineering, e.g. of the urinary bladder, and could replace organ biopsy for autologous primary cell retrieval.

CONCLUSIONS:

D., Cayan

University

OF

ESTROGEN RECEPTOR BLOCKER, FUNCTIONS AND HISTOLOGY CYSTITIS MODEL

S., Tek M., Abidinoglu

of Mersin

School

D., Akta? S., Bozlu

of Medicine,

Department

TAMOXIFEN IN A RAT

M., Akbay

of Urology,

E

Mersin,

INTRODUCTION & OBJECTIVES: Interstitial cystitis can cause progressively significant decreases in bladder capacity and compliance. High incidence of morbidity of medical and surgical treatment may limit long-term patient compliance in patients with chronic inflammation of the bladder. The purpose of this study was to investigate the effect of tamoxifen citrate on bladder functions and histology in a rat interstitial cystitis model. MATERIAL & METHODS: The study included 32 female Sprague-Dawley rats with interstitial cystitis induced by intravesical instillation of hydrochloric acid. The acid instillation was repeated monthly to maintain chronic inflammation. The treatment group (n=15) received 0.4 mg/kg/day of tamoxifen citrate with orogastric tube, and the control group (n=17) received no treatment. Urodynamic studies were performed in all rats before the treatment and at sacrifice. The rats were sacrificed at 2 weeks, 1 month and 2 months. The bladders were removed and examined histologically for mast cells and inflammatory changes. RESULTS: The maximal bladder capacity and compliance after the treatment with tamoxifen citrate significantly increased from pre-treatment to post-treatment (~~0.02 and p=O.Ol, respectively). The mean bladder capacity increased by 66.4% i24.6 in the treatment group and 21 .l% 117.7 in the control group, revealing no significantly difference (p=O. 14). The mean bladder compliance increased by 73.6% h24.7 in the treatment group and 13.5% 116.2 in the control group, revealing statistically significantly difference between the two groups (p=O.O4). At 2 weeks, the mean maxima1 bladder capacity increased by 89.2% *44.38 in the treatment group and decreased by 18.7% +13.93 in the control group, revealing statistically significantly difference (p=O.O4). The histologic studies revealed no significant differences in mast cell counts and leukocyte infiltration between the treatment and control groups. CONCLUSIONS: In this rat interstitial cystitis model, tamoxifen citrate provided functional improvement. Thus, tamoxifen citrate may be an alternative choice, as easy, to other treatment options in the treatment of chronic inflammatory condition to improve deteriorated bladder function. The next step would be to investigate the role of estrogen receptors in the diseased bladders.

627

626 ARE GONADAL ANDROGENS LOGY OF POSTMENOPAUSAL Cavzermes A.‘, Benoit

L.‘, Brailly G.‘, Droupy

S.2, Mishrahi S.’

IMPLICATED IN THE PHYSIOPATHOSTRESS URINARY INCONTINENCE? M.2, Giuliano

F.‘, Bensadoun

H.3, Jardin

‘CHU Kremlin Bic&tre, Urology, Kremlin Bic&tre, France; ‘CHU Kremlin BicCtre, Biochemistry, Kremlin BicCtre, France, XHU Caen, Urology, Caen, France INTRODUCTION & OBJECTIVES: The severity of stress urinary incontinence (WI) is not improved by using substitutive postmenopausal hormone therapy (estrogen and progesterone). Total circulating androgen decrease results in part from ovarian failure in postmenopausal women. In a first study on menopausal animal model, we found that androgen decrease result in loss of AR immunopositive nuclei staining of motoneurons innervating two muscles implicated in urinary continence. It particularly results in somal size decrease of motoneurons innervating the Pubococcygeus muscle which is implicated in stress urinary incontinence in women. To determine the role of androgens in post menopausal stress urinary incontinence, we measured the concentrations of adrenal and gonadal (sDHEA, Testosterone total and free, D4androstendione) androgens in peripheral blood of postmenopausal women. MATERIAL & METHODS: 50 postmenopausal women were included in the study. 16 did not have any urinary incontinence symptoms whether 34 suffered for SUI. The means androgens concentrations from each group were compared, and androgens levels were analysed in correlation with age in each group. To evaluate if a different androgen receptors sensibility was present between the two groups, we analysed the CAG repeats in the genomic DNA from each patient and we compared the means length between each group. RESULTS: No significant differences were found between the two groups on means androgens concentrations and means length of CAG repeats. In incontinent women gonadal androgens decreased with age with a significant correlation @=0.025). This correlation was not found in the continent group or with adrenal androgens. The length of CAG repeats was one sequence shorter in the continent group than in the incontinent group. CONCLUSIONS: Our results suggest that androgens could be implicated in the physiopathology of postmenopausal urinary incontinence. In stress urinary incontinent group androgens levels decrease with age and androgen receptors sensitivity is probably lower in women with SUI.

Turkey

EXCRETION NORMAL Maroclo

OF URINARY GLYCOSAMINOGLYCAN MENSTRUAL CYCLE OF YOUNG WOMEN

M., Pereira

State University Brazil

S., Sampaio

F. >-Cardoso

of Rio de Janeiro,

Urogenital

DURING

THE

L. Research

Unity,

Rio de Janeiro,

INTRODUCTION & OBJECTIVES: Glycosaminoglycans (GAG) play an important protective role on the urothelial surface, and changes in urinary GAG excretion have been demonstrated in several female urological diseases, especially interstitial cystitis. However, published data on GAG excretion in this disease are not consistent. This may be due to an eventual variation of GAG excretion during the menstrual cycle, since controls consisted of women at childbearing age. Here we used well-established biochemical techniques and a homogenous study group to assess GAG excretion profile during the menstrual cycle of normal young women. MATERIAL & METHODS: Urine samples were obtained from 10 medical students not meeting exclusion criteria (irregular menstrual cycles, use of oral contraceptives, urinary infections, history of urolithiasis and diabetes) and aged 19 to 21 years. Each volunteer collected one urine specimen daily during the whole menstrual cycle. Total GAG was isolated from diluted urine by precipitations with cetylpyridinium chloride and ethanol, and was then quantitated as hexuronic acid using a carbazole assay. The urinary concentration of GAG was expressed as pg hexuronic acid per mg creatinine. Proportions of sulfated GAG species were determined by agarose gel electrophoresis. RESULTS: GAG excretion showed a biphasic pattern, with higher and lower values in the pre- and post-ovulatory periods, respectively. In addition, mean excretion values obtained for the periods between the 4th and 13th (0.445 * 0.041), and between the 15th and 28th (0.356 i- 0.035) days of the cycle were significantly different (one-way ANOVA, p
Urology

Supplements

3 (2004)

No. 2, pp. 159