Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and...

Area of Science:

• Biotechnology
• Tissue Engineering
• Biomedical Engineering

Background:

• Dynamic culture environments are crucial for bioengineered skeletal muscle tissues, improving nutrient delivery and waste removal.
• Perfusion systems offer enhanced physiological biomimicry and advanced analytical capabilities for in vitro tissues.
• Perfusion systems must accommodate multiple cell-laden constructs for diverse applications.

Purpose of the Study:

• To report on perfusion systems manufactured using additive manufacturing for in situ myogenic precursor cell development.
• To assess the biocompatibility and performance of 3D printed perfusion systems for tissue engineering.
• To investigate the effects of perfusion on cellular development and gene expression in skeletal muscle models.

Main Methods:

• Additive manufacturing techniques including stereolithography (SL), laser sintering (LS), and PolyJet were used to produce perfusion systems.
• Biocompatibility was assessed by evaluating cell morphology within the 3D printed devices.
• Myogenic precursor cells (C2C12) in monolayer and bioengineered tissues were cultured under intermittent and continuous perfusion for up to 13 days.
• Gene expression analysis (MyoD, myogenin, MyHC, IL-1β, TNF-α, myostatin, MuRF-1) and live cellular imaging were performed.

Main Results:

• SL and LS 3D printed devices showed preferential morphological development.
• Intermittent perfusion in monolayer cultures resulted in delayed but physiologically representative cellular proliferation and gene transcription.
• Continuous perfusion of bioengineered tissues led to in situ myogenic differentiation and formation of multinucleated myotubes.
• Perfusion cultures showed reduced inflammatory cytokines (IL-1β, TNF-α), myostatin, and MuRF-1 mRNA expression.
• While MyHC isoform profiles were comparable, total mRNA expression was reduced in perfusion conditions, suggesting decreased MyHC gene expression requirements.

Conclusions:

• 3D printed perfusion systems, particularly those from SL and LS, are biocompatible and support myogenic development.
• Perfusion culture enhances the stability of the culture environment for bioengineered skeletal muscle.
• Perfusion culture reduces the basal requirement for MyHC gene expression in mature bioengineered skeletal muscle tissue.