title: Factors affecting the structure and maturation of human tissue engineered skeletal muscle.
abstract: Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 × 10(5) MDCs assembled to a greater extent than those at 1 × 10(5) or 2 × 10(5), and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS(®) technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue.
authors: Musculoskeletal Biology Research Group, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire LE113TU, UK.
authors: Neil R W
authors: NR
authors: Martin
authors: Samantha L
authors: SL
authors: Passey
authors: Darren J
authors: DJ
authors: Player
authors: Alastair
authors: A
authors: Khodabukus
authors: Richard A
authors: RA
authors: Ferguson
authors: Adam P
authors: AP
authors: Sharples
authors: Vivek
authors: V
authors: Mudera
authors: Keith
authors: K
authors: Baar
authors: Mark P
authors: MP
authors: Lewis
mesh_terms: D000328:Adult; D000368:Aged; D002452:Cell Count; D002459:Cell Fusion; D005260:Female; D005337:Fibrin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018189:Immunomagnetic Separation; D008297:Male; D008875:Middle Aged; D024510:Muscle Development / Q000187:drug effects / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds; D055815:Young Adult
chemical_list: D012333:RNA, Messenger; D005337:Fibrin; D018995:Myosin Heavy Chains