Shah, R.; (2010) The in vitro engineering of craniofacial muscle constructs utilising degradable composite glass fibre-collagen scaffolds. Doctoral thesis , UCL (University College London).

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Abstract

Absent or defective craniofacial skeletal muscle can lead to loss of function and aesthetics. Current therapies are fraught with limitations: for example, the surgical transfer of tissue is associated with donor site morbidity coupled with a paucity of available tissue. The potential to engineer skeletal muscle tissue could circumvent disadvantages related to current techniques. Furthermore, the creation of a muscle testbed could provide the means to investigate the response to various manipulations. The aim of this research was to produce an in vitro human craniofacial skeletal muscle tissue suitable as a test-bed for novel therapies involving the craniofacial region. Degradable phosphate-based glass fibre scaffolds of various configurations, combined with extracellular matrix (ECM) components, were seeded with human craniofacial muscle-derived cell cultures. Myogenicity was confirmed with immunofluorescent techniques prior to seeding. The seeded scaffolds were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for up to 21 days. Modulation contrast microscopy was used to analyse migration and morphology. Cell attachment and survival were assessed with the CyQUANT® and alamarBlue® assays, and cell differentiation and maturation were investigated using immunofluorescence and quantitative RT-PCR. Parallel arrays of glass fibres coated with ECM components provided the correct topology to support cell alignment and differentiation. Specifically, compared to control scaffolds, glass fibre scaffolds promoted upregulation of developmental, fast and slow myosin heavy chain genes. Further refinement of the system involved glass fibres embedded within collagen gels, created to mimic the architecture of native skeletal muscle: cells within these constructs were aligned parallel to the glass fibres, and over time, the constructs rolled along the short axis to produce a muscle ‘organoid’. Additionally, the collagen gel contracted along the long axis to reveal tufts of glass fibres analogous to tendons (mean 13.87% reduction in length). Upregulation of the myosin heavy chain genes was promoted, albeit at a later timepoint. In conclusion, degradable glass fibre-ECM scaffolds provided the correct topographical and biological cues to aid the in vitro engineering of human craniofacial skeletal muscle tissue.

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