Functional regeneration of tissue engineered skeletal muscle in vitro is dependent on the inclusion of basement membrane proteins

Abstract

Skeletal muscle has a high regenerative capacity, injuries trigger a regenerative program which restores tissue function to a level indistinguishable to the pre-injury state. However, in some cases where significant trauma occurs, such as injuries seen in military populations, the regenerative process is overwhelmed and cannot restore full function. Limited clinical interventions exist which can be used to promote regeneration and prevent the formation of non-regenerative defects following severe skeletal muscle trauma. Robust and reproducible techniques for modelling complex tissue responses are essential to promote the discovery of effective clinical interventions. Tissue engineering has been highlighted as an alternative method, allowing the generation of three-dimensional in vivo like tissues without laboratory animals. Reducing the requirement for animal models promotes rapid screening of potential clinical interventions, as these models are more easily manipulated, genetically and pharmacologically, and reduce the associated cost and complexity, whilst increasing access to models for laboratories without animal facilities. In this study, an in vitro chemical injury using barium chloride is validated using the C2C12 myoblast cell line, and is shown to selectively remove multinucleated myotubes, whilst retaining a regenerative mononuclear cell population. Monolayer cultures showed limited regenerative capacity, with basement membrane supplementation or extended regenerative time incapable of improving the regenerative response. Conversely tissue engineered skeletal muscles, supplemented with basement membrane proteins, showed full functional regeneration, and a broader in vivo like inflammatory response. This work outlines a freely available and open access methodology to produce a cell line-based tissue engineered model of skeletal muscle regeneration.

Keywords: bioengineering; regeneration; skeletal muscle physiology; tissue engineering.

Figure 1

Treatment of differentiated C2C12 cultures with BaCl₂ specifically removes myotubes from culture and initiates a regenerative response. (a) ×20 widefield micrographs of recovery time points in C2C12s. Stained for actin (phalloidin, red) and nuclei (DAPI, blue), scale bars represent 50 μm. (b) Fusion index, (c) total nuclei, (d,e) measures of myotube maturity. Values for 0 hr and 2 days post injury have been omitted as too few myotubes were present in these conditions to accurately measure these variables (b–e) Graphs express mean ± SD, asterisks above bars denotes significance from control, ***denotes significance p < .001, (f–i) RT‐PCR analysis of cellular developmental and inflammatory markers. All graphs display mean ± SD, *denotes significance p < .05, **denotes significance p < .01, ***denotes significance p < .001. (j) Experimental timeline for generation of differentiated C2C12 culture, injury and subsequent recovery. RT‐PCR, real time‐polymerase chain reaction

Figure 2

Type I collagen hydrogels lack regenerative capacity following injury. (a) representative confocal microscope tile scans at ×40 magnification consisting of 21 individual images. Stained with phalloidin (red) and DAPI (blue) to identify actin and nuclei, respectively. Scale bars denote 250 μm. (b) Myotube density expressed as myotubes per 100 μm. Mean ± SD. (c) Total nuclei per image frame. Mean ± SD, *p < .05. (d) Experimental time course of the 28 day 3D recovery experiment

Figure 3

Inclusion of Matrigel® allows 3D culture models to support regeneration following insult from BaCl_2. (a) Representative tile scans at ×40 magnification, phalloidin staining (red) and DAPI (blue) identify actin and nuclei, respectively. Scale bars represent 100 μm. (b) Myotube density expressed as myotubes per 100 μm (solid bars) and percentage of gel occupied by myotubes (hashed bars). Mean ± SD, significance from control denoted by asterisks above bars, **p < .01, ***p < .001. (c) Nuclei per image frame. Mean ± SD. (d) Myotube width (μm). Mean ± SD, significance from control ***p < .001. (e) Force output data, twitch (1 Hz, solid bars) and fused tetanus (100 Hz, hashed bars) peak force expressed as mean ± SD, significance from control, **p < .01. (f) Representative force traces for C2C12 constructs stimulated at different frequencies to achieve; single twitch (1 Hz) and fused tetanus (100 Hz)

Figure 4

RT‐PCR analysis of gene expression during recovery of 3D tissue engineered muscle. (a–d) All graphs display means ± SD, significance from control denoted by asterisk; *p < .05, **p < .01. RT‐PCR, polymerase chain reaction