206 MODULATION OF MICROENVIRONMENT BY GROWTH FACTORS REGULATES IN VIVO GROWTH OF SKELETAL MYOBLASTS

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THE JOURNAL OF UROLOGY姞

envisioned as excellent cell sources for muscle tissue engineering with promising results in preclinical studies for the treatment of stress urinary incontinence. Optimal muscular reconstruction involves formation of new muscle fibers capable of performing tonic contractions which is only possible if the same oxidative muscle fiber type, nerve ingrowth and formation of new neuromuscular junctions (NMJ) can be induced. In this study, we evaluate whether Noninvasive electromagnetic stimulation (NMS) improves tissue engineered muscle regeneration after MPC implantation, by investigating the presence of synapses, clustering of acetylcholine receptors (AChRs) and muscular metabolic adaptations caused by muscle contraction. METHODS: MPCs were isolated from a muscle biopsy of patients undergoing abdominal surgery and implanted in vivo intramuscularly in a collagen carrier into a mouse model of muscle injury. Half of the animals were submitted to 5 consecutive days of NMS (BioCon2000, 20 min, 50 Hz). Animals that received MPCs transfected with PGC1␣, which boost muscle metabolism, as well as not stimulated animals served as controls. Muscle regeneration, nerve ingrown and formation of new NMJ were investigated by immunohistochemistry, RTPCR and WB. RESULTS: NMS caused no damage on the stimulated animals and increased stem cell differentiation in vitro and in vivo (p⬍0.001). The number of synapses (p⫽0.013), detected by acetylcholinesterase expression, and cluster of acetylcholine receptors (AChR) doubled under NMS treatment (p⫽0.003). Our analyses also revealed that muscle metabolism (p⬍0.001), measured by PGC1␣ expression, and muscle reconstruction were boosted by the NMS therapy. Nerve ingrowth was stimulated, with increase of Agrin production on stimulated muscle (p⬍0.05), and increase on nerve branching (p⬍0.05). Finally, our data show that NMS treatment significantly improved stem cells integration and distribution into the regenerating muscle (p⬍0.001), increasing the number of fibers formed by the implanted MPCs. CONCLUSIONS: NMS improves differentiation of implanted MPCs into well organized fibers with molecular features of normal muscle, by improving synapses, and boosting nerve ingrown and AChR clustering. Source of Funding: Hartmann-Mu¨ller Foundation, Forschunskredit of University of Zu¨rich

206 MODULATION OF MICROENVIRONMENT BY GROWTH FACTORS REGULATES IN VIVO GROWTH OF SKELETAL MYOBLASTS Akihiro Yanagiuchi, Hideaki Miyake*, Masato Fujisawa, Kobe, Japan INTRODUCTION AND OBJECTIVES: With advances in the field of tissue engineering, transplantation of myogenic stem cells, called myoblasts, have been extensively investigated as a therapeutic option in muscle-related diseases. Such an approach seems to be extremely promising for the treatment of stress urinary incontinence, since myoblasts have the ability to undergo differentiation and fusion to form myofibers, leading to sphincter regeneration; however, to date, transplantation of myoblasts by direct intramuscular injection in vivo have shown limited efficiency in the muscle integration of donor cells. The objective of this study was to investigate the optimal microenvironment for efficient myoblast transplantation in vivo focusing on the interaction between myoblasts and growth factors. METHODS: The effects of coculture with growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I) and platelet-derived growth factor (PDGF), on in vitro growth, migration and proteolytic activity of mouse skeletal myoblasts were investigated. Myoblasts were coinjected with growth factors into the subcutis and bladder wall of nude mice, and its impact on the growth patterns of myoblasts in vivo was assessed. RESULTS: Dose-dependent stimulation of in vitro myoblast growth was observed following treatment with each of the four kinds of growth factors, but bFGF induced the most remarkable increase in the

Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012

growth of myoblasts. Treatment of myoblasts with all kinds of growth factors also resulted in a dose-dependent increase in the in vitro migration of myoblasts, and PDGF exerted the most prominent effect on myoblast migration. Increased secretion of matrix metalloproteinase-9 (MMP-9) in myoblasts induced by growth factors was proportional to their increased migration capacity, which was partially inhibited by SB-3CT, a specific inhibitor of MMP-9. The in vivo growth of myoblasts was significantly enhanced by coinjection with all kinds of growth factors into both the subcutis and bladder wall; however, this effect was most remarkable 1 and 2 weeks after coinjection with bFGF and PDGF, respectively. Furthermore, synergistic in vivo growth of myoblasts was observed by coinjection of both bFGF and PDGF compared with that achieved with either agent alone. CONCLUSIONS: These findings suggest that modulation of the microenvironment using growth factors, particularly bFGF and PDGF, could provide the optimal condition for efficacious myoblast transplantation in vivo. Source of Funding: None

207 AN INNOVATIVE APPROACH TO BUILDING A CLINICALLY RELEVANT SIZED TISSUES AND ORGANS FOR RECONSTRUCTION Jaehyun Kim*, Tanner Hill, Sang Jin Lee, James Yoo, Anthony Atala, Winston Salem, NC INTRODUCTION AND OBJECTIVES: Building a clinically relevant sized tissue or organ using cells requires maintenance of viable cells until host vasculature is established and integrated into the implanted engineered constructs. However, delay in vasculogenesis results in premature cell death due to the inadequate supply of oxygen and nutrients. One potential solution is to develop methods to maintain cell viability over a long-term by downregulating cellular metabolism until host vascularization is established. Adenosine, a nucleoside which functions as an energy transferring molecule, is reported to increase during hypoxia and functions as a modulator of ion-channel arrest. In this study, we attempted to promote cell survival under hypoxic conditions by exploiting the property of adenosine. METHODS: Myoblasts (C2C12 cells) designated as the hypoxic group were transferred to the hypoxic chamber (0.1% O2). A group with no adenosine was placed under hypoxia for up to 13 days to demonstrate eventual cell death. Another group receiving daily doses of adenosine (0 to 10 mM) was incubated for up to 7 days under hypoxia and then placed back into normoxic conditions. The metabolic activity of viable cells at each pre-determined time point was assessed using an MTS assay, which measures mitochondrial activity of cells. RESULTS: The metabolic activity of cells grown in normoxia increased linearly with respect to time. Hypoxic cells not treated with adenosine showed an increasing metabolic activity for 7 days under hypoxia, but this resulted in eventual cell death. However, when treated with adenosine, cells under hypoxic conditions maintained a steady state of metabolic activity and these cells resumed their normal metabolic activity instantly when normoxic conditions were restored and adenosine was removed at 7 days. As the dose of adenosine increased from 0 to 10 mM, an escalation of steady hypometabolic state was maintained under hypoxic conditions, and the cells resumed their normal metabolic activity after 7 days. CONCLUSIONS: We demonstrate the novel concept that cell viability can be maintained by downregulating cellular metabolism under hypoxic conditions. Application of adenosine to cells under hypoxic conditions prolongs survival by decreasing the metabolic activity to a steady hypometabolic state, thus reducing O2 demand. This concept represents a novel method for increasing cellular survival in tissue-engineered constructs during vasculogenesis. Source of Funding: This study was supported, in part, by a grant from the Department of Defense (W81XWH-07-1-0718).