Article

Engineering of aligned skeletal muscle by micropatterning

American Journal of Translational Research (Impact Factor: 3.4). 01/2010; 2(1):43-55.
Source: PubMed

ABSTRACT

Tissue engineered skeletal muscle has tremendous potential for the treatment of muscular injury or muscular dysfunction. However, in vitro methods to generate skeletal muscle with physiologically aligned myofiber structure remains limited. To develop a robust in vitro model that resembles the physiologically aligned structure of muscle fibers, we fabricated micropatterned polymer membranes of poly(dimethylsiloxane) (PDMS) with parallel microgrooves, and then examined the effect of micropatterning on myoblast cellular organization and the cell fusion process. In comparison to the myoblasts on non-patterned PDMS films, myoblasts on micropatterned PDMS films had well-organized F-actin assembly in close proximity to the direction of microgrooves, along with enhanced levels of myotube formation at early time points. The increase of cell cycle regulator p21(WAF/Cip1) and the organized interactions of N-cadherin in myoblasts on micropatterned surfaces may contribute to the enhanced formation of myotubes. Similar results of cellular alignment was observed when myoblasts were cultured on microfluidically patterned poly(2-hydroxyethyl methacrylate) (pHEMA) microgrooves, and the micropatterns were found to detach from the Petri dish over time. To apply this technology for generating aligned tissue-like muscle constructs, we developed a methodology to transfer the aligned myotubes to biodegradable collagen gels. Histological analysis revealed the persistence of aligned cellular organization in the collagen gels. Together, these results demonstrate that micropatterned PDMS or pHEMA can promote cell alignment and fusion along the direction of the microgrooves, and this platform can be utilized to transfer aligned myotubes on biodegradable hydrogels. This study highlights the importance of spatial cues in creating aligned skeletal muscle for tissue engineering and muscular regeneration applications.

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    • "In vitro, cell elongation, alignment, and function can be controlled using anisotropic features from nano-(Biela et al., 2009; Hamilton et al., 2010; Teixeira et al., 2003; Yim et al., 2005) or micro-grooved (Clark et al., 1987, 1990; Dalton et al., 2001; Oakley and Brunette, 1995a; Oakley et al., 1997; Wood et al., 2012) substrates. Grooves typically produce either whole-cell alignment (Biela et al., 2009; Britland et al., 1996; Brunette, 1986; Cimetta et al., 2009; den Braber et al., 1996a; Diehl et al., 2005; Fujita et al., 2006; Grew et al., 2008; Hamilton et al., 2010; Huang et al., 2010; Loesberg et al., 2007; McDevitt et al., 2003; Munir et al., 2011; Oakley and Brunette, 1995a; Poole et al., 2005; Su et al., 2007; Sutherland et al., 2005; Truong et al., 2010; Tsai et al., 2009; Wang et al., 2011) or cytoskeletal alignment (Biela et al., 2009; Uttayarat et al., 2008) and groove depth, as opposed to width or spacing, is the critical parameter for determining the extent of alignment. Specifically groove depths must be above a certain threshold value to elicit a response, typically in the range of 35–250 nm (Biela et al., 2009; Clark et al., 1990; Loesberg et al., 2007). "
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    ABSTRACT: Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial, umbilical vein endothelial, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible we have also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: i) the ability of neighbouring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighbouring cells in flat regions over a limited distance of approximately 200 microns. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues. © 2014 Wiley Periodicals, Inc.
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    ABSTRACT: Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies.
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