Engineered Myoblast-Laden Collagen Filaments Fabricated Using a Submerged Bioprinting Process to Obtain Efficient Myogenic Activities.

Abstract

The skeletal muscle tissue comprises a hierarchical fibrous structure with fully aligned myofibers. To obtain a unique aligned engineering construct for regenerating muscle tissue, we adopted a submerged bioprinting process. Here, 3 wt % collagen and 6 wt % alginate solutions were used as a matrix cell-encapsulating bioink and supporting solution in the printing bath, respectively. By manipulating the processing parameters (various alginate weight fractions in the bath, nozzle moving speed, and hydrostatic pressure), cell-laden filaments (∼50 μm in diameter) were successfully fabricated. They presented a high degree of alignment of the fibrillated collagen and meaningful initial viability (∼90%) of the C2C12 myoblasts. In vitro cellular responses indicated that fully aligned F-actin filaments of myoblasts were developed, resulting in a high degree of alignment/formation of myotubes, compared to that in the controls (>100 μm diameter of cell-laden filaments). Furthermore, the expression levels of various myogenic genes (Myod1, Myh2, and Myog) were measured using a reverse transcription polymerase chain reaction on day 21 of the cell culture, and the results showed that the cell-laden filaments with a small diameter had considerably greater gene expression levels (2.2-8-fold) than those with a relatively large diameter. Thus, the printing process described herein can provide a new potential biofabricating platform to obtain cell-laden engineering constructs for various tissues.