The in vitro engineering of craniofacial muscle constructs utilising degradable composite glass fibre-collagen scaffolds

摘要

Absent or defective craniofacial skeletal muscle can lead to loss of function andudaesthetics. Current therapies are fraught with limitations: for example, the surgicaludtransfer of tissue is associated with donor site morbidity coupled with a paucity ofudavailable tissue. The potential to engineer skeletal muscle tissue could circumventuddisadvantages related to current techniques. Furthermore, the creation of a muscle testbedudcould provide the means to investigate the response to various manipulations. Theudaim of this research was to produce an in vitro human craniofacial skeletal muscle tissueudsuitable as a test-bed for novel therapies involving the craniofacial region.udDegradable phosphate-based glass fibre scaffolds of various configurations, combinedudwith extracellular matrix (ECM) components, were seeded with human craniofacialudmuscle-derived cell cultures. Myogenicity was confirmed with immunofluorescentudtechniques prior to seeding. The seeded scaffolds were incubated at 37°C in audhumidified atmosphere of 5% CO2 in air for up to 21 days. Modulation contrastudmicroscopy was used to analyse migration and morphology. Cell attachment andudsurvival were assessed with the CyQUANT® and alamarBlue® assays, and celluddifferentiation and maturation were investigated using immunofluorescence andudquantitative RT-PCR.udParallel arrays of glass fibres coated with ECM components provided the correctudtopology to support cell alignment and differentiation. Specifically, compared to controludscaffolds, glass fibre scaffolds promoted upregulation of developmental, fast and slowudmyosin heavy chain genes. Further refinement of the system involved glass fibresudembedded within collagen gels, created to mimic the architecture of native skeletaludmuscle: cells within these constructs were aligned parallel to the glass fibres, and overudtime, the constructs rolled along the short axis to produce a muscle ‘organoid’.udAdditionally, the collagen gel contracted along the long axis to reveal tufts of glassudfibres analogous to tendons (mean 13.87% reduction in length). Upregulation of theudmyosin heavy chain genes was promoted, albeit at a later timepoint.udIn conclusion, degradable glass fibre-ECM scaffolds provided the correct topographicaludand biological cues to aid the in vitro engineering of human craniofacial skeletal muscleudtissue.