Speaker
Description
Introduction: Despite the crucial role of the muscle extracellular matrix in the organotypic organization and the transmission of mechanical force, most 3D muscle models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy and porosity. In vivo, muscle extracellular matrix possesses specific characteristics such as a high amount of aligned collagen I to create an anisotropic structure with a significant porosity and a suitable stiffness. Recent approaches of muscle models used non porous hydrogels fabricated from low concentrated collagen to encapsulate muscle cells. Hence, the in vivo properties are not reproduced. Here, we developed a 3D printed collagen hydrogel that mimic the muscle extracellular matrix, i.e collagen anisotropy, adequate stiffness and two ranges of porosity (one to ensure nutrients and oxygen diffusion and the other for cell cultivation).
Methodology: Dense collagen solutions (30 mg.ml-1) were printed through the 23G flat bottom needle inside a buffer bath. The extrusion process aligned the collagen molecules along the axis of extrusion. The buffer bath played two major roles: it “froze” the collagen alignment and triggered collagen gelling. The printing process was performed unidirectionally for each layer to create an intrinsic porosity between the different collagen filaments. After a rapid period of collagen gelling, needles were introduced within the hydrogel to generate large pores. An additional gelling period was performed to tune the mechanical properties. C2C12, murine skeletal muscle cells were then seeded within the printed hydrogels to evaluate the cell colonization, myotube formation and organotypic organization.
Results: By tuning the extrusion speed and the gelling process, a 3D printed hydrogel with aligned collagen fibers was obtained. A combination of two gelling strategies (24h PBS 5X + 24h NH3) was optimal to obtain both anisotropy and adequate mechanical properties (E=10 kPa). Scaffold anisotropy was obtained at two different scales: all filaments were printed in the same direction (macroscopic) and collagen fibers were aligned inside printed filaments (microscopic). Concerning the porosity, changing the height between two successive layers allowed to create an intrinsic porosity from 50 to 150 µm. Interestingly, the generation of 100 µm pores preserved the scaffold cohesiveness. This porosity is suitable for nutrients and oxygen diffusion in the whole scaffold, thereby favoring cell viability. Larger pores created by needles molding generated straight channels of 600 µm in diameter. These were easily colonized by C2C12 cells mixed with Matrigel® to create a suitable 3D environment. After 4 days of differentiation, aligned multinucleated myotubes were formed. Immunostaining with sarcomeric heavy chain myosin revealed the cell commitment into mature myotubes.
Conclusions: In this study, we developed a 3D printing technique to create a biomimetic muscle extracellular matrix suitable for muscle cell differentiation and cultivation. Our approach focused on the extracellular matrix and its key parameters since it is deeply involved in muscular functions. Hence, this model could be used with patients cells to study and have a better understanding on muscular dystrophies.
20941802324
