Application of 3D Printing Technology in Skeletal Muscle Vascularization Research

Academic Background

Skeletal muscle tissue is one of the most important tissues in the human body, and its function relies on the close relationship between myotubes and the vascular network. The vascular network not only supplies oxygen and nutrients to skeletal muscle but also plays a key role in muscle injury repair. However, when skeletal muscle injury exceeds 20%, the effectiveness of traditional autologous muscle transplantation is limited, with a high failure rate. In recent years, advancements in tissue engineering have provided new possibilities for skeletal muscle repair, particularly through the application of three-dimensional (3D) printing technology, which has made it possible to construct skeletal muscle tissue with vascular networks. However, how to precisely regulate the interaction between skeletal muscle and the vascular network in vitro remains a significant challenge.

This study aims to explore the complex interactions between skeletal muscle and endothelial cells during vascularization using 3D bioprinting technology and to develop a method for precise temporal and spatial regulation of skeletal muscle vascularization. By constructing biomimetic vascularized skeletal muscle tissue, the research team hopes to provide new insights for future tissue engineering and regenerative medicine.

Source of the Paper

This paper was co-authored by Minxuan Jia, Tingting Fan, Tan Jia, Xin Liu, Heng Liu, and Qi Gu. The research team is affiliated with the Institute of Zoology, Chinese Academy of Sciences, the Beijing Institute for Stem Cell and Regenerative Medicine, and Beijing Jishuitan Hospital, Capital Medical University. The paper was published online on September 26, 2024, in the journal Bio-design and Manufacturing, with the DOI 10.1007/s42242-024-00315-0.

Research Process and Results

1. Cell Culture and Bioink Preparation

The research team began by culturing mouse myoblast cell line C2C12 and human umbilical vein endothelial cells (HUVECs). C2C12 cells were cultured in proliferation medium, while HUVECs were grown in gelatin-coated dishes. For 3D printing, the team prepared two types of bioinks: one composed of fibrinogen and gelatin loaded with C2C12 cells, and the other a fibrinogen/gelatin mixture (HFG) loaded with HUVECs.

2. 3D Printing and Skeletal Muscle Construction

The research team used a 3D bioprinter to print C2C12 cell-laden bioink into skeletal muscle bundles. During the printing process, temperature and pressure were adjusted to ensure the bioink remained in a gel state. After printing, the skeletal muscle bundles were fixed to pre-printed scaffolds and crosslinked with thrombin. Experimental results showed that the width of the printed muscle bundles changed significantly during the initial culture period, but the cell viability remained as high as 90%, indicating minimal impact of 3D printing on the cells.

3. Construction of Vascularized Skeletal Muscle

To construct vascularized skeletal muscle, the research team designed two strategies: one involved adhering a monolayer of HUVECs to the surface of the skeletal muscle bundles, and the other involved wrapping HFG around the exterior of the muscle bundles. The experiments revealed that the monolayer-adhered HUVECs gradually detached during culture, failing to form a stable vascular network. In contrast, the HFG-wrapping method allowed HUVECs to be cultured long-term on the exterior of the muscle bundles, gradually forming a vascular network. Additionally, the team found that introducing HUVECs during the later stages of skeletal muscle differentiation significantly promoted myotube formation and vascular network development.

4. Experimental Results and Analysis

Using immunofluorescence staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR), the research team evaluated the differentiation and vascularization levels of the vascularized skeletal muscle. The results showed that in the group where HUVECs were introduced on day 7 of skeletal muscle differentiation (DM7+6 group), the length and nuclear count of myotubes increased significantly, indicating more mature differentiation. Furthermore, the expression of vascularization markers CD31 and VE-cadherin was significantly higher in this group, suggesting more robust vascular network formation.

Conclusions and Significance

This study successfully constructed biomimetic vascularized skeletal muscle tissue using 3D bioprinting technology. The results demonstrate that introducing endothelial cells during the later stages of skeletal muscle differentiation significantly promotes myotube formation and vascular network development. This finding provides new insights for future tissue engineering and regenerative medicine, particularly in the construction of large-scale, physiologically aligned skeletal muscle.

Research Highlights

1. Innovative Methodology: This study is the first to combine 3D bioprinting with 3D modeling technology to achieve co-construction of skeletal muscle and vascular networks.
2. Temporal Regulation: By precisely controlling the timing of vascular network introduction, the research team successfully optimized the skeletal muscle differentiation process.
3. Biomimetic Properties: The constructed vascularized skeletal muscle tissue exhibits high biomimetic properties, laying the foundation for future clinical applications.

Other Valuable Information

The research team also developed a “three-in-one mold” for wrapping HFG around the exterior of skeletal muscle bundles. This mold, made of polydimethylsiloxane (PDMS), has excellent flexibility and hydrophobicity, effectively reducing fibrin adhesion and facilitating operation and demolding.

This study not only provides a new research method for skeletal muscle vascularization but also opens new avenues for future tissue engineering and regenerative medicine.