A three‐dimensional in vitro model system to study the adaptation of craniofacial skeletal muscle following mechanostimulation

Abstract

The aim of this study was to investigate the in vitro response of human craniofacial muscle-derived myotubes (primitive/nascent muscle fibres), in three-dimensional constructs, to strain in vitro to mimic clinical scenarios, using expression of the mechanoresponsive gene gelatinase-A/matrix metalloproteinase-2 (MMP-2) as a marker of remodelling of muscle extracellular matrix. Three-dimensional (3D) constructs of cells derived from explants of human masseter muscle (human craniofacial muscle-derived cells; hCMDC) in collagen sponges were subjected to mechanical, uniaxial strain using the Bio-Stretch system. 3D myotube constructs were exposed to the strain regimes of rapid ramp stretch (RRS) or cyclical ramp strain (CRS) with 7.5% and 15% strain. The activity of MMP-2 was assessed by zymography of construct-conditioned medium, whilst lysates of the constructs were used to measure creatine phosphokinase (CPK) activity to confirm the presence of myotubes in the strained constructs. Scanning electron microscopy of the collagen sponges and the CPK assays confirmed the presence of myotubes. MMP-2 was expressed by all the samples and controls, but expression was found to be significantly higher in those cultures strained continuously (RRS), compared to cyclical strain (CRS), and in those strained at 15% compared to 7.5%. Thus, MMP-2 expression, and hence extracellular matrix remodelling, is up-regulated in response to strain and is dependent upon the amount and type of strain to which the muscle is subjected.

Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts

“…Moreover, biomechanical forces such as tension, compression and shear have been shown to modulate the activity of MMP-2 in chondrocytes (Fujisawa et al, 1999), ligaments (Zhou et al, 2005), muscle (O'Callaghan and Williams, 2000;Auluck et al, 2005) and in the cardiovascular system (Milkiewicz et al, 2005;Rastogi et al, 2005).…”

A novel in vitro three-dimensional skeletal muscle model

“…Some examples of this type of research include (1) the effects of compression, tension, and contraction on skeletal muscle constructs (Okano and Matsuda 1998;Dennis and Kosnik 2000;Bouten et al 2003;Cheema et al 2003;Auluck et al 2005), (2) effects of hypoxia on viability (Gawlitta et al 2007), (3) metabolic activities (Baker et al 2003), (4) effects of culture conditions (electrical impulses, conditioned medium; Pedrotty et al 2005), and (5) co-culture of muscle cells with other cell types (Larkin et al 2006;Murshid et al 2007). However, despite the availability of a variety of existing models and constructs, skeletal muscle differentiation and regeneration require further definition (Charge and Rudnicki 2004;Horsley and Pavlath 2004).…”

Stretching skeletal muscle in vitro: does it replicate in vivo physiology?

“…Similarly, using the 3D collagen system, MMP-2 activity increased following stretching in a load-dependent manner (Auluck et al 2005).…”

“…A number of 3D biomimetic model systems have been described with the intention of recreating aspects of the 3D environment and so promoting the formation of physiological muscle models. Such models include the use of collagen gel matrices (Powell et al 2002;Cheema et al 2003), or collagen gel foam sponges (Auluck et al 2005) to provide a physiologically relevant matrix into which the muscle cells are seeded. Muscle constructs grown in such a 3D environment under uniaxial strain have been shown to exhibit a slow phenotype analogous to that of postural muscle in vivo (Mudera et al 2010).…”