Tissue Engineering Approaches for the Craniofacial Deformity Patient

Objectives: The craniofacial soft tissue defect is notoriously difficult to manage. Tissue engineering has the potential to provide a suitable tissue for implantation. The study objectives were to (1) engineer 3D skeletal muscle constructs in vitro; (2) utilize a customized bioreactor to exert force to the muscle constructs; (3) investigate response of the skeletal muscle extracellular matrix to external force as an indication of muscle adaptation.
Methods: C2C12 myoblasts were seeded at 2x10⁶ cells/ml within 3D collagen constructs and maintained at 37^○C in 5% CO₂ in air for 7 days to allow for myofiber formation. The engineered skeletal muscle constructs were then subjected to 10% ramped cyclical strain (n=3) or fixed strain (n=3) within a customized bioreactor for 5 days. The control group (n=3) was not subjected to external force. Microscopy and RT-PCR determined myogenicity. ELISA determined expression of matrix metalloproteinases (MMP) 2 and 9, and tissue inhibitors of matrix metalloproteinases (TIMP) 1 and 2, as an indication of muscle extracellular matrix (ECM) response to external force.

Results: Upregulation of the myogenic regulatory factors, MyoD1 and myogenin, confirmed myogenicity. ELISA demonstrated MMP-2 expression to be significantly greater than MMP-9, TIMP-1 and TIMP-2 expression (P<0.05) in all groups indicating a shift towards ECM degradation. The skeletal muscle constructs subjected to external force demonstrated greater MMP and TIMP expression compared to the control group (P<0.05). The increase and relative proportions of MMPs and TIMPs suggested greater muscle ECM adaptation within constructs subjected to ramped cyclical strain as compared to fixed strain.
Conclusions: Customized bioreactors can house engineered 3D skeletal muscle constructs long-term. External force leads to greater muscle ECM degradation and by extrapolation adaptation to new functional demands. Utilization of external force could improve on engineered skeletal muscle tissue prior to implantation. Further research will determine the clinical impact.