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Bioengineering strategies to enhance myoglobin expression in 3D C2C12 muscle constructs
Citation Link: https://doi.org/10.15480/882.17649
Publikationstyp
Doctoral Thesis
Date Issued
2026
Sprache
English
Author(s)
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2026-06-04
Institute
TORE-DOI
Citation
Technische Universität Hamburg (2026)
Developing physiologically relevant in vitro models of skeletal muscle tissue remains a central and persistent challenge across tissue engineering, regenerative medicine, and cultivated meat research. A critical barrier to achieving both scalability and advanced function is the absence of a simple, cost-effective fabrication strategy, coupled with insufficient clarity regarding the control mechanisms governing tissue maturation and key metabolic parameters like oxygenation. Myoglobin, the crucial oxygen-binding protein in muscle, serves as a vital functional biomarker for metabolic capacity and tissue maturity. This thesis directly addresses these fundamental obstacles through three integrated studies. First, a novel, scaffold-free protocol was successfully developed for fabricating three-dimensional (3D) skeletal muscle constructs with good structural integrity from C2C12 myoblasts. This innovative method leverages a spontaneous delamination phenomenon induced solely by serum starvation, offering a distinct advantage in simplicity and cost-effectiveness over conventional techniques. The resulting constructs were characterized for high viability, structural integrity determined via histology and scanning electron microscopy, and successful myogenic differentiation confirmed through myosin heavy chain expression. Second, to overcome inherent scalability limitations, computational modeling was integrated computational modeling with meticulous experimental validation to estimate a critical size constraint for these dense, cell-only constructs. Finite element simulations, validated by experimental measurements, established a maximum viable thickness of 5 mm based on the model's prediction that oxygen concentration at the construct's core would drop to zero at this boundary. This constraint is fundamentally driven by the inherent biophysical characteristics of the engineered tissue, thus establishing an important, data-informed design parameter for all future upscaling efforts. Finally, a a broad range of biochemical and physical parameters was systematically investigated to enhance myoglobin protein synthesis, the central indicator of tissue maturation and coloration. The findings showed that myoglobin synthesis is not solely driven by supplementation with iron, creatine, or Insulin-like Growth Factor-I (IGF-I), but is instead profoundly regulated by the physical and chemical microenvironment. Specifically, culture vessel size was inversely correlated with myoglobin content, with smaller vessels yielding significantly higher levels. Controlled bioreactor experiments identified a moderate dissolved oxygen (DO) concentration of 30 % air saturation as most favorable for myoglobin synthesis. Furthermore, applying a cyclic mechanical stretching regimen, when combined with optimal DO, induced a synergistic increase in myoglobin levels, resembling physiological responses to muscle exercise. Altogether, this work presents an integrated framework to creating, optimizing, and functionally enhancing scaffold-free muscle constructs. Four critical parameters, culture vessel size, culture time, dissolved oxygen levels, and mechanical stimulation, were identified that collectively govern myoglobin synthesis. These findings provide a useful foundation for developing physiologically relevant engineered muscle constructs with potential applications, from disease modeling to sustainable food production.
Subjects
Myoglobin
Muscle tissue engineering
3D Construct
Oxygen transfer simulation
C2C12
Cultivated Meat
DDC Class
610: Medicine, Health
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Name
KhozaeiRavari_Mojtaba_Bioengineering Strategies to Enhance Myoglobin Expression in 3D C2C12 Muscle Constructs.pdf
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