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2018 ; 6
(ä): e4939
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3D skeletal muscle fascicle engineering is improved with TGF-?1 treatment of
myogenic cells and their co-culture with myofibroblasts
#MMPMID30018850
Krieger J
; Park BW
; Lambert CR
; Malcuit C
PeerJ
2018[]; 6
(ä): e4939
PMID30018850
show ga
BACKGROUND: Skeletal muscle wound healing is dependent on complex interactions
between fibroblasts, myofibroblasts, myogenic cells, and cytokines, such as
TGF-?1. This study sought to clarify the impact of TGF-?1 signaling on skeletal
muscle cells and discern between the individual contributions of fibroblasts and
myofibroblasts to myogenesis when in co-culture with myogenic cells. 3D
tissue-engineered models were compared to equivalent 2D culture conditions to
assess the efficacy of each culture model to predictively recapitulate the in
vivo muscle environment. METHODS: TGF-?1 treatment and mono-/co-cultures
containing human dermal fibroblasts or myofibroblasts and C2C12 mouse myoblasts
were assessed in 2D and 3D environments. Three culture systems were compared:
cell monolayers grown on 2D dishes and 3D tissues prepared via a self-assembly
method or collagen 1-based hydrogel biofabrication. qPCR identified gene
expression changes during fibroblast to myofibroblast and myoblast
differentiation between culture conditions. Changes to cell phenotype and tissue
morphology were characterized via immunostaining for myosin heavy chain,
procollagen, and ?-smooth muscle actin. Tissue elastic moduli were measured with
parallel plate compression and atomic force microscopy systems, and a slack test
was employed to quantify differences in tissue architecture and integrity.
RESULTS: TGF-?1 treatment improved myogenesis in 3D mono- and co-cultures
containing muscle cells, but not in 2D. The 3D TGF-?1-treated co-culture
containing myoblasts and myofibroblasts expressed the highest levels of myogenin
and collagen 1, demonstrating a greater capacity to drive myogenesis than
fibroblasts or TGF-?1-treatment in monocultures containing only myoblasts. These
constructs possessed the greatest tissue stability, integrity, and muscle fiber
organization, as demonstrated by their rapid and sustained shortening velocity
during slack tests, and the highest Young's modulus of 6.55 kPA, approximate half
the stiffness of in situ muscle. Both self-assembled and hydrogel-based tissues
yielded the most multinucleated, elongated, and aligned muscle fiber histology.
In contrast, the equivalent 2D co-culture model treated with TGF-?1 completely
lacked myotube formation through suppression of myogenin gene expression.
DISCUSSION: These results show skeletal muscle regeneration can be promoted by
treating myogenic cells with TGF-?1, and myofibroblasts are superior enhancers of
myogenesis than fibroblasts. Critically, both TGF-?1 treatment and co-culturing
skeletal muscle cells with myofibroblasts can serve as myogenesis accelerators
across multiple tissue engineering platforms. Equivalent 2D culture systems
cannot replicate these affects, however, highlighting a need to continually
improve in vitro models for skeletal muscle development, discovery of
therapeutics for muscle regeneration, and research and development of in vitro
meat products.