title: 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.
authors.affiliation: Orthodontic Unit, Eastman Dental Institute, University College London, 256 Grays Inn Road, London WC1 8LD, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Auluck
authors.forename: Vivek
authors.initials: V
authors.lastname: Mudera
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Hunt
authors.forename: Mark P
authors.initials: MP
authors.lastname: Lewis
mesh_terms: D000222:Adaptation, Physiological / Q000502:physiology*; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000032:analysis; D005109:Extracellular Matrix / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D005781:Gelatin Sponge, Absorbable / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008406:Masseter Muscle / Q000502:physiology / Q000648:ultrastructure*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000502:physiology* / Q000648:ultrastructure; D009940:Organoids / Q000201:enzymology / Q000648:ultrastructure; D010812:Physical Stimulation; D013314:Stress, Mechanical; D015854:Up-Regulation
chemical_list: D015415:Biomarkers; D003402:Creatine Kinase; D020778:Matrix Metalloproteinase 2