Brady, M.; (2008) Mechanical or biological cues?: Parameters to engineer a 3D human masseter muscle. Doctoral thesis , University of London.

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Abstract

Skeletal muscle lends itself to the central tenet of tissue engineering in that myoblasts isolated from skeletal muscle tissue are able to recapitulate the exact processes that occur in vitro during muscle development (Shah et al., 2005). In vitro skeletal muscle engineering involves the culture of isolated primary myogenic cells in an environment conducive to the formation of a three-dimensional (3D) tissue construct, containing differentiated myotubes, capable of generating force. The aim of this thesis was to test the effect of mechanical and biological cues on the development of a tissue engineered craniofacial masseter muscle construct. For the first time primary myogenic, non myogenic and heterogenous (mixture of myogenic and non-myogenic) cells were tested in a 3D collagen scaffold to define parameters for in vitro engineering of human masseter muscle. The cytomechanical properties of the human masseter muscle derived cells (MDCs) in 3D were quantified over 24 hours using a culture force monitor (CFM) and the results showed a synergistic response, the heterogenous mixture generated significantly (p<0.01) more force than the sum of the individual components of separated cells. Further, muscle specific (myogenin, myosin heavy chain (MYHC)) and matrix remodeling (MMP-2) gene expressions were upregulated relative to the separated individual cell types. To study mechanical (cell-matrix) cues MDCs were subject to external loading and increased matrix stiffness. The results showed no significant change in gene expression. To study biological (cell-cell) cues, MDCs were cultured at high densities and for an extended period of time (6 days). The results showed an upregulation of muscle specific (myogenin, MYHC-IIX/D, MYHC-P) gene expression. However, MMP-2 gene expression remained unchanged. These results indicated that cell-cell signals are more important than cell-matrix signals in 3D myogenic cell differentiation. This study highlights the importance of non-myogenic cells in force generation and the effect of increasing substrate stiffness and cell density in control of construct maturation. These are the first parameters defined towards successful 3D human muscle engineering.

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