Introduction: Skeletal muscle, one of the most abundant tissues in the human body, is essential for vital physiological functions and organ movements. It serves as the primary driver of bodily motion. Despite its impressive regenerative ability, significant injuries leading to volumetric muscle loss (VML) overwhelm the natural regenerative mechanisms. When the tissue loss exceeds 15-20% of the total muscle volume, functional recovery becomes unattainable. This creates a pressing need for innovative approaches in skeletal muscle tissue engineering to restore muscle function and improve patient outcomes. Traditional methods like casting have been used to engineer skeletal muscle tissue. However, they fall short in replicating the complex structure and cellular organization required for effective muscle function. As a solution, 3D bioprinting emerges as a promising technique. It offers precise control over the spatial distribution of cells and biomaterials, enabling the creation of constructs that closely mimic native muscle tissue. This study leverages embedded bioprinting, an innovative approach that remains relatively unexplored for muscle tissue engineering, to fabricate a rectangular muscle ring. We focus on optimizing various parameters to enhance the structural and functional properties of the engineered muscle. By refining the bioprinting process, we aim to produce muscle constructs with improved cellular organization and viability, advancing the capabilities of muscle tissue engineering.
Materials and
Methods: We used a custom-made extrusion-based 3D bioprinter to fabricate the muscle rings. The bioink composition included fibrinogen at concentrations ranging from 1 to 8 mg/ml, with alginate added for enhanced stability and printability at concentrations of 0.5 to 1.5% (wt/v). Thrombin concentration was varied from 4 to 20 U/ml. The support bath was composed of Laponite-RDS nanoclay, Pluronic F127, calcium chloride, and thrombin. The support bath was optimized in a previous study to ensure proper hydrogel formation during printing. Various concentrations of calcium chloride and thrombin in the support bath were tested to further refine its crosslinking and rheological properties. Different cell densities of C2C12 myoblasts, ranging from 1 to 5 x 106 cells/ml, were used to study cell distribution within the bioprinted rings. Different cell densities of C2C12 myoblasts were used to study cell distribution within the bioprinted rings. The bioprinted muscle rings were incubated at 37°C to allow crosslinking of the hydrogels, with incubation times ranging from 30 minutes to 1.5 hours. After incubation, the bioprinted structures were carefully washed to remove the support bath, ensuring the stability and integrity of the muscle rings for further analysis.
Results, Conclusions, and Discussions: Optimization of the support bath composition, including 0.25% calcium chloride and 20 U/ml thrombin, significantly enhanced the printability and structural integrity of the muscle rings. Rheological analysis revealed that the support bath exhibited ideal viscosity and recoverability, critical for maintaining construct shape and stability during and after printing. Additionally, cyclic strain measurements demonstrated the support bath's ability to recover quickly over a short time range, further ensuring its suitability for bioprinting applications. Bioprinting of muscle rings within the optimized support bath yielded constructs with promising characteristics. Muscle tissue engineering requires very high cell densities compared to other 3D bioprinted tissues, making this a challenging yet significant achievement. High cell density is crucial for muscle tissue to ensure sufficient cell-cell interactions and to promote the formation of functional muscle fibers. The distribution of C2C12 myoblasts was uniform across various cell densities, with higher concentrations resulting in improved cellular organization and viability. After recovery from the support bath, the bioprinted muscle rings maintained their structural integrity. The constructs, composed of 6 mg/ml fibrinogen and 1.5% alginate, displayed signs of significant cellular proliferation after three days in growth media. This suggests the potential for cell viability, proliferation, and migration within the constructs. Such observations provide valuable insights into the initial stages of muscle tissue development using bioprinting techniques. As a future perspective, our objective is to further characterize cell distribution and cell alignment throughout the bioprinted structure and spatially implement magnetic particles via 3D printing to control intercellular mechanical stimulation more precisely. This advancement could offer new avenues for enhancing tissue maturation and functionality in bioprinted muscle constructs.
