title: Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.
abstract: Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.
authors.affiliation: School of Biomedical Science, CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia.
authors.forename: Vishal
authors.initials: V
authors.lastname: Chaturvedi
authors.affiliation: School of Biomedical Science, CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia.
authors.forename: Danielle E
authors.initials: DE
authors.lastname: Dye
authors.affiliation: School of Biomedical Science, CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia.
authors.forename: Beverley F
authors.initials: BF
authors.lastname: Kinnear
authors.affiliation: Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.affiliation: School of Anatomy, Physiology and Human Biology, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Miranda D
authors.initials: MD
authors.lastname: Grounds
authors.affiliation: School of Biomedical Science, CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia.
authors.forename: Deirdre R
authors.initials: DR
authors.lastname: Coombe
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D021982:Cell-Matrix Junctions; D024141:Collagen Type IV / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D054501:Group II Phospholipases A2 / Q000378:metabolism; D008810:Mice, Inbred C57BL; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000166:cytology*; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000378:metabolism*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D054457:Tissue Scaffolds
chemical_list: D024141:Collagen Type IV; D016895:Culture Media, Serum-Free; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D054501:Group II Phospholipases A2