Pelvic organ prolapse, the decent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with mechanical alterations of the vaginal wall. In order to characterize the complex mechanical behavior of the vagina, we performed planar biaxial tests of vaginal specimens in both the passive (relaxed) and active (contracted) states. Specimens were isolated from virgin, female Long-Evans rats (n=16) and simultaneously stretched along the longitudinal direction (LD) and circumferential direction (CD) of the vagina. Tissue contraction was induced by electric field stimulation (EFS) at incrementally increasing values of stretch and, subsequently, by KCl. On average, the vagina was stiffer in the CD than in the LD (p<0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p<0.001). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p<0.01). When comparing the mean maximum EFS-induced active stress to the mean KCl-induced active stress, no differences were found in the CD (p=0.404) but, in the LD, the mean active stress was much higher in response to the KCl stimulation (p<0.001). Collectively, these results demonstrate that the anisotropic behavior of the vaginal tissue is determined not only by the collagen and smooth muscle fiber organization but also by the innervation. The findings of this study may contribute to the development of more effective treatments for pelvic organ prolapse. / MS / Pelvic organ prolapse (POP), the decent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with alterations of the mechanical properties of the vaginal wall. The characterization of the mechanical properties of the vagina is crucial for the development of effective treatments for POP. Biaxial tensile tests were performed in this study so we could observe the behavior of the vagina along both the circumferential direction (CD) and the longitudinal direction (LD). In these tests, square specimens were secured along all four edges and pulled outward such that we could observe the relationship between the stretch and the stress that the tissue experienced. Additionally, because the vagina contains smooth muscle, we also tested the tissue in its active, or contractile state at each stretch level. Contractions were induced by applying electric field stimulation (EFS) to observe nerve-mediated responses, and subsequently by potassium chloride (KCl). On average, the vagina was stiffer in the CD than in the LD (p<0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p<0.001). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p<0.01).

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12877号 / 論農博第2804号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4876(農学部図書室) / 31595 / (主査)教授 伏木 亨, 教授 保川 清, 教授 安達 修二 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM