Effect of Sustained Tension on Bladder Smooth Muscle Cells in Three-Dimensional Culture TIFFANY ROBY,SHAWN OLSEN, and JIRO NAGATOMI Department of Bioengineering, Clemson University, 501 Rhodes Engineering Research Center, Clemson, SC 29634-0905, USA (Received 5 December 2007; accepted 28 July 2008) Abstract—Previous studies demonstrated that bladder cells respond to changes in their mechanical environments by exhibiting alterations in cellular functions, such as hypertro- phy or fibrosis. In the present study, we hypothesize that changes in smooth muscle cell (SMC) behavior triggered by mechanical stimuli may represent a phenotypic shift between contractile and synthetic phenotypes. Using a custom-made device, rat bladder SMCs were cultured in three-dimensional (3-D) collagen gels and exposed to sustained tension. When compared to no-tension controls, SMCs exposed to tension exhibited significantly (p < 0.05) higher expression of alpha- smooth muscle actin (a-SMA), while cell population density was similar in both groups. In addition, both mean and median aspect ratios of SMCs in 3-D collagen constructs exposed to tension were significantly (p < 0.05) greater than those of cells cultured under no externally applied tension, indicating that there are more elongated, spindle-shaped cells in the tension group. These SMCs in 3-D cultures exposed to tension also exhibited cellular alignment along the direction of applied tension. Since contractile SMCs are known to exhibit greater expression of phenotypic marker proteins as well as a more elongated morphology, we concluded that sustained tension on cells is an important mechanical stimulus for maintenance of the contractile phenotype of bladder SMCs in vitro. Keywords—Bladder outlet obstruction, Smooth muscle cell, Phenotype, Sustained tension, Cell morphology. INTRODUCTION Previous in vivo studies have demonstrated that abnormal mechanical environments in the bladder associated with various pathological conditions trigger cellular and molecular changes, such as smooth muscle cell (SMC) hyperplasia or hypertrophy and alteration of the extracellular matrix (ECM). 10,13 These cellular and ECM changes can, in turn, deteriorate the func- tion of the bladder by decreasing the contractility and compliance of the tissue. 13 The clinical impacts of these in vivo results motivated the development of numerous in vitro studies that examined the responses of bladder SMCs to applied mechanical stimuli, such as cyclic stretch. 15–17 More specifically, it has been reported that compared to static controls, bladder SMCs exposed to cyclic stretch (0.1 Hz, max 25% stretch, up to 48 h) exhibited an increase in DNA synthesis mediated by increased synthesis and release of growth factors. 15–17 These changes in bladder SMC behavior, especially the increased proliferation, in response to stretch suggest that bladder SMCs exhibit a change from a contractile to a synthetic phenotype under certain pathological con- ditions. Contractile and synthetic SMCs represent two opposite ends of the phenotypic spectrum with a continuum of varying phenotypic intermediates, with SMCs of varying phenotypes expressing different lev- els of marker proteins rather than different proteins altogether. 19 In the vascular biology literature, soluble biochemical factors, ECM components, and physical factors have all been reported to affect the expression of SMC phenotypic marker proteins. 19,22 Previous studies in the urology literature have also implicated a change in bladder SMC phenotype in response to cyclic stretch, which was demonstrated by decreased alpha-smooth muscle actin (a-SMA) organization, increased prolif- eration, and increased secretion of ECM proteins. 5,24 Although these studies provided some valuable information, they share a common method of two- dimensional (2-D) culture of SMCs, which does not completely represent physiological conditions since the bladder cells in vivo exist as clusters surrounded by collagen sheaths in a three-dimensional (3-D) environment. 20 The overall goal of the present study, therefore, is to develop a 3-D culture of bladder SMCs which will allow investigation of the effects of mechanical stimuli on SMC phenotype within the 3-D environment. More specifically, in the present study, we examined the effects of sustained uni-directional Address correspondence to Jiro Nagatomi, Department of Bio- engineering, Clemson University, 501 Rhodes Engineering Research Center, Clemson, SC 29634-0905, USA. Electronic mail: jnagato@ clemson.edu Annals of Biomedical Engineering, Vol. 36, No. 10, October 2008 (Ó 2008) pp. 1744–1751 DOI: 10.1007/s10439-008-9545-5 0090-6964/08/1000-1744/0 Ó 2008 Biomedical Engineering Society 1744