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MYOBLASTS CAN FORM NEW MUSLE TISSUE AND INDUCE ANGIO- AND NEUROGENESIS
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THE JOURNAL OF UROLOGY®
37.3 ± 3.3 cmH2O). Maximal steady state contractile responses of isolated tissue strips from regenerating bladders decreased following STC, but also demonstrated functional recovery. Immunohistochemistry revealed progenitor cells positive for c-kit (CD117) in the detrusor, and positive for p63 in the urothelium. PGP9.5 positive cells were seen 4 weeks post-STC illustrating reinnervation. Immunohistochemistry also revealed mature Uroplakin 3 (UP3) positive umbrella cells as early as 1 week post-STC. CONCLUSIONS: We used high resolution imaging modalities and direct measures of bladder function in vivo in combination with in vitro measures of tissue pharmacology and histology to characterize de novo bladder regeneration. This approach may provide noninvasive markers of physiological milestones as well as the cellular events critical to bladder regeneration. The functional duration of the regenerated bladder is not known. The long-term goal is to leverage the novel insights obtained to maximize the bladder’s regenerative capacity/potential for clinical applications. Source of Funding: NIH
210 MYOBLASTS CAN FORM NEW MUSLE TISSUE AND INDUCE ANGIO- AND NEUROGENESIS Dr. Hannes Strasser*, Innsbruck, Austria INTRODUCTION AND OBJECTIVE: Myoblasts have been used in experimental and clinical studies for decades. It was the aim of the present study to investigate the potential of myoblasts after injection to create new muscle tissue and induce angio- as well as neurogenesis. METHODS: To create a closed compartment without migration and inflluence of adjacent cells, myoblasts were mixed with collagen (Contigen®, bard, USA) as carrier material and injected into a polyethersulfon tube that was permeable for trophic factors but impermeable for cells in 20 Fisher rats. The tube was implanted into the region of the right groin. A motoric branch of the femoral nerve as well as the accompanying vessels were then implanted into the tube, and the tube was then closed. The PES tube containined only implanted cells as well as the vesel and verve branch. 8 weeks after ímplantation the whole tube was removed. The whole tube was then analysed histologically to investigate the formation of new muscle tissue, angiogenesis as well as neurogenesis. RESULTS: It could be demonstrated that the that young myoblasts freshly outgrown in vitro on a three-dimensional carrier structure could create new muscle fibers and muscle tissue. Most striking, a huge amount of new and small blood vessels that were located not around but between the muscle fibers could be observed after implantation of the cells, forming a dense three-dimensional network of blood supply. Neurofilament-specific staining showed that newly fomed nerve fibers had sprouted from the nerve branch. By means of cholinesteraseevidence-reaction it was possible to show that new motoric nerve endplates had formed. Monoclonal antibodies against bungarotoxin, having a high affinity to acetylcholin receptors, showed similar positive results in fluorescence microscopy. CONCLUSIONS: The pesent data show that injected myoblasts form new muscle tissue under the influence of motoric innervation. There is a close and strong interaction between the injected myoblasts and adjacent vessels and nerves. Injected myoblasts have a high potential to induce angio- and neurogenesis, which is substantial for differentiation and the the physiologic properties of the newly formed musle tissue. Moreover, the presence of new motoric endplates shows that the new muscle and nerve fibers are brought into contact. This interaction and formation of new muscle tissue and nerves is not dependent on surrounding cells and anatomic structures, as this model of a closed compartment shows. This is also true for angiogenesis. Source of Funding: Innovacell Grant
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
211 THE EFFECT OF LAMINA PROPRIA CELLS ON THE GROWTH OF UROTHELIAL AND SMOOTH MUSCLE CELLS Roberto Soler*, Winston Salem, NC; Claudius Fullhase, Munich, Germany; Nadia Guimaraes-Souza, Karl-Erik Andersson, James J Yoo, Winston Salem, NC INTRODUCTION AND OBJECTIVE: Epithelial-mesenchymal interactions are mediated, in part, by paracrine and juxtacrine factors, which are believed to be secreted by the cells residing in the lamina propria (LP). Recently, several biological growth factors were identified in the porcine bladder lamina propria matrix. In our attempt to grow lamina propria (LP) cells in culture, we observed that these cells exhibited a high level of proliferation. So, we designed a study to evaluate the effect of LP cells on the proliferation of urothelial cells (UC) and smooth muscle cells (SMC). METHODS: UC, SMC and LP cells were isolated from their respective tissue layers from porcine bladders (n=4) using established harvesting techniques. Cell populations from each source were culture expanded in their growth media supplemented with 10% fetal bovine serum (SMC; Dulbecco’s Modified Eagles Medium, (DMEM), UC; keratinocyte growth medium supplemented with 5 ng/ml of epidermal growth factor and 50 g/ml of bovine pituitary extract, LP cells; mixture of both mediums at 1:1 ratio). The cultured cell populations were characterized using cell specific antibodies. UC and/or SMC were grown under three different conditions: 1) cultured over the mitomycin C treated LP cell feeder layer to assess juxtacrine effects, 2) cultured in the presence of LP cells (2x104) grown on a 1μm porous membrane to assess paracrine effects, and 3) grown on regular tissue culture plates (control). Cells were trypsinized on days 2, 4 and 6 and counted using a hemocytometer. RESULTS: Both the UC and SMC proliferated rapidly in the presence of LP cells on the porous membrane. On day 4, the number of UC was significantly higher in the co-culture with LP cells when compared to the control cells (p=0.018). On day 6, the mean number of UC in the co-culture was almost the double of the control (p=0.04). A similar proportion was observed in the mean number of SMC co-cultured with LPC in comparison to its control on day 6 (p=0.017). Similar growth patterns were observed in conditions where UC and SMC were grown over the LP cell feeder layer CONCLUSIONS: LP cells accelerated the growth of UC and SMC in vitro. We showed that LP cells are able to influence cellular proliferation either through cell-cell (juxtacrine) or factor mediated (paracrine) interactions. Therefore, for being a natural source of biological active factors, the use of LP cells may be an interesting strategy in cell culture and tissue engineering of the bladder. Further investigation is necessary to identify factors that control UC and SMC development and differentiation. Source of Funding: None
212 A LYMPHATIC VESSEL NETWORK IN UROTHELIUM OF THE URINARY BLADDER Kazumasa Matsumoto*, Shigehiro Soh, Takefumi Satoh, Erina Satoh, Tetsuo Fujita, Sagamihara, Japan; Yukio Ishikawa, Toshiharu Ishii, Tokyo, Japan; Masatsugu Iwamura, Masatsugu Iwamura, Shiro Baba, Sagamihara, Japan INTRODUCTION AND OBJECTIVE: Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is a novel lymphatic vessel marker that is expressed on lymph vessel endothelial cells. LYVE-1 was not only identified as a valuable marker of lymphatic endothelial cells in normal human tissues, but also succeeded in immunohistochemical staining for small lymphatic vessels in bladder tissues. However, the distribution of lymphatic vessels and the density of benign tissues in the urinary bladder still remain to be found. The objective of this study was to determine LYVE-1 expression patterns in normal urothelium and to compose the geometric topography of the lymphatic network. METHODS: Immunohistochemical staining for LYVE-1 and von Willebrand factor was carried out and assessed the differences in distribution of lymphatic vessels between the components in the urinary bladder. The
THE JOURNAL OF UROLOGY®
37.3 ± 3.3 cmH2O). Maximal steady state contractile responses of isolated tissue strips from regenerating bladders decreased following STC, but also demonstrated functional recovery. Immunohistochemistry revealed progenitor cells positive for c-kit (CD117) in the detrusor, and positive for p63 in the urothelium. PGP9.5 positive cells were seen 4 weeks post-STC illustrating reinnervation. Immunohistochemistry also revealed mature Uroplakin 3 (UP3) positive umbrella cells as early as 1 week post-STC. CONCLUSIONS: We used high resolution imaging modalities and direct measures of bladder function in vivo in combination with in vitro measures of tissue pharmacology and histology to characterize de novo bladder regeneration. This approach may provide noninvasive markers of physiological milestones as well as the cellular events critical to bladder regeneration. The functional duration of the regenerated bladder is not known. The long-term goal is to leverage the novel insights obtained to maximize the bladder’s regenerative capacity/potential for clinical applications. Source of Funding: NIH
210 MYOBLASTS CAN FORM NEW MUSLE TISSUE AND INDUCE ANGIO- AND NEUROGENESIS Dr. Hannes Strasser*, Innsbruck, Austria INTRODUCTION AND OBJECTIVE: Myoblasts have been used in experimental and clinical studies for decades. It was the aim of the present study to investigate the potential of myoblasts after injection to create new muscle tissue and induce angio- as well as neurogenesis. METHODS: To create a closed compartment without migration and inflluence of adjacent cells, myoblasts were mixed with collagen (Contigen®, bard, USA) as carrier material and injected into a polyethersulfon tube that was permeable for trophic factors but impermeable for cells in 20 Fisher rats. The tube was implanted into the region of the right groin. A motoric branch of the femoral nerve as well as the accompanying vessels were then implanted into the tube, and the tube was then closed. The PES tube containined only implanted cells as well as the vesel and verve branch. 8 weeks after ímplantation the whole tube was removed. The whole tube was then analysed histologically to investigate the formation of new muscle tissue, angiogenesis as well as neurogenesis. RESULTS: It could be demonstrated that the that young myoblasts freshly outgrown in vitro on a three-dimensional carrier structure could create new muscle fibers and muscle tissue. Most striking, a huge amount of new and small blood vessels that were located not around but between the muscle fibers could be observed after implantation of the cells, forming a dense three-dimensional network of blood supply. Neurofilament-specific staining showed that newly fomed nerve fibers had sprouted from the nerve branch. By means of cholinesteraseevidence-reaction it was possible to show that new motoric nerve endplates had formed. Monoclonal antibodies against bungarotoxin, having a high affinity to acetylcholin receptors, showed similar positive results in fluorescence microscopy. CONCLUSIONS: The pesent data show that injected myoblasts form new muscle tissue under the influence of motoric innervation. There is a close and strong interaction between the injected myoblasts and adjacent vessels and nerves. Injected myoblasts have a high potential to induce angio- and neurogenesis, which is substantial for differentiation and the the physiologic properties of the newly formed musle tissue. Moreover, the presence of new motoric endplates shows that the new muscle and nerve fibers are brought into contact. This interaction and formation of new muscle tissue and nerves is not dependent on surrounding cells and anatomic structures, as this model of a closed compartment shows. This is also true for angiogenesis. Source of Funding: Innovacell Grant
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
211 THE EFFECT OF LAMINA PROPRIA CELLS ON THE GROWTH OF UROTHELIAL AND SMOOTH MUSCLE CELLS Roberto Soler*, Winston Salem, NC; Claudius Fullhase, Munich, Germany; Nadia Guimaraes-Souza, Karl-Erik Andersson, James J Yoo, Winston Salem, NC INTRODUCTION AND OBJECTIVE: Epithelial-mesenchymal interactions are mediated, in part, by paracrine and juxtacrine factors, which are believed to be secreted by the cells residing in the lamina propria (LP). Recently, several biological growth factors were identified in the porcine bladder lamina propria matrix. In our attempt to grow lamina propria (LP) cells in culture, we observed that these cells exhibited a high level of proliferation. So, we designed a study to evaluate the effect of LP cells on the proliferation of urothelial cells (UC) and smooth muscle cells (SMC). METHODS: UC, SMC and LP cells were isolated from their respective tissue layers from porcine bladders (n=4) using established harvesting techniques. Cell populations from each source were culture expanded in their growth media supplemented with 10% fetal bovine serum (SMC; Dulbecco’s Modified Eagles Medium, (DMEM), UC; keratinocyte growth medium supplemented with 5 ng/ml of epidermal growth factor and 50 g/ml of bovine pituitary extract, LP cells; mixture of both mediums at 1:1 ratio). The cultured cell populations were characterized using cell specific antibodies. UC and/or SMC were grown under three different conditions: 1) cultured over the mitomycin C treated LP cell feeder layer to assess juxtacrine effects, 2) cultured in the presence of LP cells (2x104) grown on a 1μm porous membrane to assess paracrine effects, and 3) grown on regular tissue culture plates (control). Cells were trypsinized on days 2, 4 and 6 and counted using a hemocytometer. RESULTS: Both the UC and SMC proliferated rapidly in the presence of LP cells on the porous membrane. On day 4, the number of UC was significantly higher in the co-culture with LP cells when compared to the control cells (p=0.018). On day 6, the mean number of UC in the co-culture was almost the double of the control (p=0.04). A similar proportion was observed in the mean number of SMC co-cultured with LPC in comparison to its control on day 6 (p=0.017). Similar growth patterns were observed in conditions where UC and SMC were grown over the LP cell feeder layer CONCLUSIONS: LP cells accelerated the growth of UC and SMC in vitro. We showed that LP cells are able to influence cellular proliferation either through cell-cell (juxtacrine) or factor mediated (paracrine) interactions. Therefore, for being a natural source of biological active factors, the use of LP cells may be an interesting strategy in cell culture and tissue engineering of the bladder. Further investigation is necessary to identify factors that control UC and SMC development and differentiation. Source of Funding: None
212 A LYMPHATIC VESSEL NETWORK IN UROTHELIUM OF THE URINARY BLADDER Kazumasa Matsumoto*, Shigehiro Soh, Takefumi Satoh, Erina Satoh, Tetsuo Fujita, Sagamihara, Japan; Yukio Ishikawa, Toshiharu Ishii, Tokyo, Japan; Masatsugu Iwamura, Masatsugu Iwamura, Shiro Baba, Sagamihara, Japan INTRODUCTION AND OBJECTIVE: Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is a novel lymphatic vessel marker that is expressed on lymph vessel endothelial cells. LYVE-1 was not only identified as a valuable marker of lymphatic endothelial cells in normal human tissues, but also succeeded in immunohistochemical staining for small lymphatic vessels in bladder tissues. However, the distribution of lymphatic vessels and the density of benign tissues in the urinary bladder still remain to be found. The objective of this study was to determine LYVE-1 expression patterns in normal urothelium and to compose the geometric topography of the lymphatic network. METHODS: Immunohistochemical staining for LYVE-1 and von Willebrand factor was carried out and assessed the differences in distribution of lymphatic vessels between the components in the urinary bladder. The