Bioprinting Perfusable and Vascularized Skeletal Muscle Flaps for the Treatment of Volumetric Muscle Loss

Immunology Biochemistry Life Sciences Cell Biology Bioengineering Cardiovascular System Medicine Basic Medical Sciences
bioprinting vascularization skeletal muscle flaps volumetric muscle loss tissue engineering perfusion transplantation

Academic Report on “Bioprinting Perfusable and Vascularized Skeletal Muscle Flaps for the Treatment of Volumetric Muscle Loss”

Background

Muscle tissues constitute a significant portion of human cellular mass and are a complex, highly vascularized, and dynamic tissue. However, traumatic or surgical Volumetric Muscle Loss (VML)—defined as the loss of over 20% of muscle tissue in a functional area—often causes severe functional disabilities. Standard treatment methods primarily rely on autologous muscle flap transfers from a healthy donor site to the damaged area, but such surgeries frequently lead to donor site morbidity and are limited by the constrained availability of muscle tissues.

Currently, the field of tissue engineering (TE) focuses on developing new methods based on cells and extracellular matrix (ECM) to regenerate muscles and restore functionality. However, traditional techniques like using acellular matrices or cellular sheets often lack structural stratification and vascularization, which limits implant viability due to diffusion constraints. While existing studies have explored the use of 3D bioprinting to create vascularized muscle grafts, these technologies predominantly depend on lengthy decellularization processes (Decellularized ECM Bioinks) and lack stratified vascular structures capable of rapid perfusion.

This study introduces a novel multimodal bioprinting approach that allows for the fabrication of thick vascularized muscle flaps with hierarchical vascular networks and engineered macrovessels. These innovations enable in vivo muscle flap implantation, immediate perfusion, and integration, offering a potential solution to diffusion bottlenecks and paving the way for new VML treatment modalities.

Source and Research Team

The article titled “Bioprinting Perfusable and Vascularized Skeletal Muscle Flaps for the Treatment of Volumetric Muscle Loss” was conducted by Eliana O. Fischer, Anna Tsukerman, Shulamit Levenberg, and their team at the Faculty of Biomedical Engineering, Technion-Israel Institute of Technology. The research was published in the Advanced Healthcare Materials journal in 2025 and was funded by the European Union’s Horizon 2020 research and innovation program (Project No. 818808).

Research Process Detailed Analysis

The study, focused on multimodal 3D bioprinting technology, adopted a stepwise design to complete the process from in vitro printing to in vivo implantation. Below is an overview of the primary steps:

1. Optimization of Bioink and Cells

  • Subjects: The study utilized bioinks derived from human-origin cells, including Human Skeletal Muscle Cells (HSKMCs), Human Umbilical Vein Endothelial Cells (HUVECs), and pericytes.
  • Bioink Preparation: A fibrin-based bioink was prepared, consisting of fibrinogen (15 mg/ml) and thrombin (10 U/ml), with cell densities as high as 35–40 × 10⁶ cells/ml.
  • Preliminary Tests: Plug tests were conducted to evaluate the vascularization and muscle differentiation properties under monoculture (HSKMCs), co-culture (HUVECs + Pericytes), and tri-culture (HSKMCs + HUVECs + Pericytes) conditions. Fluorescence intensity and viability analyses demonstrated survival rates exceeding 90% across all groups.

2. Bioprinting Stratified Vascular Structures

  • Bioprinting Process:
    • Using extrusion bioprinting, skeletal muscle cells, endothelial cells, and support cells were sequentially printed to create composite tissues.
    • A macrovessel made of liquid PLLA/PLGA was embedded around the printed fibrin gels to prevent structural collapse.
  • Formation of Hierarchical Vascular Network: The macrovessel was connected to branched microvessels and the printed muscle tissue, creating a mechanically stable and uniformly distributed vascular network.

3. Fluid Dynamics Modeling and Mechanical Testing of Vascular Structure

  • Computational Fluid Dynamics (CFD): Simulations using ANSYS software examined flow velocities, shear stress, and pressure in both macrovessels and microvessels. Results indicated alignment with the natural flow parameters of rat femoral arteries.
  • Finite Element Analysis (FEA): The mechanical properties of the macrovessel were defined by an elastic modulus of 40.5 MPa and a safety factor of 15, confirming its resistance under physiological pressures.

4. Constructing Functional Systems and In Vivo Implantation

  • Functional Validation: The engineered macrovessel was sutured via microsurgical techniques to the rat femoral artery for immediate perfusion. Perfusion efficiency was verified with Laser-Speckle Analysis.
  • Control Groups:
    • Experimental Group: Perfused bioprinted muscle flaps.
    • Positive Control: Non-perfused muscle grafts without arterial manipulation.
    • Negative Control: Femoral artery ligation.
    • The perfused flap group maintained blood flow rates above 65%, while the femoral artery ligation group showed only 50% of normal limb blood flow.

Research Results in Detail

1. Vascular Perfusion and Functional Validation

  • Micro-CT Imaging: Perfusion within the engineered muscle flaps showed clear contrast agent distribution in the lumen of the macrovessel, indicating patent blood flow.
  • Histological Analysis: CD31 staining and H&E results showed abundant neovascularization and dense red blood cell distribution in the perfused group.

2. Muscle Differentiation and Tissue Integration

  • Microscopic Imaging: Newly formed muscle fibers in the perfused group exhibited alignment and significant muscle differentiation markers (Desmin, Myosin Heavy Chain).
  • Myonuclear Evaluations: Muscle cell nuclei counts were significantly higher in the perfused group, with an average of approximately 39.2 nuclei per fiber.
  • Tissue Activity Indicators: The perfused group demonstrated higher matrix density, with minimal hyaluronic acid deposition.

3. Mechanical Performance and Perfusion Behavior

  • The macrovessel maintained shape and continuity under physiological pressures, while flow dynamics in microvessels aligned closely with natural vascular parameters.

Conclusions and Significance of the Research

Findings: This study marks the first successful development of implantable, perfused multimodal 3D bioprinted vascularized muscle flaps. These flaps demonstrated immediate perfusion and integration with the circulatory system, significantly boosting neovascularization, muscle differentiation, and tissue viability.

Significance: 1. Scientific Value: The production of efficient stratified vascularized muscle constructs has broad implications for tissue regeneration and organ engineering by overcoming traditional diffusion limitations. 2. Clinical Applications: This approach provides a transformative pathway for customizable large-scale tissue repair and VML treatment.

Highlights: - Innovative multimodal bioprinting strategy. - Physiologically simulated and efficient vascular networks. - Rapid muscle differentiation, reducing in vitro incubation time.

Summary

This study envisions a new perspective for treating VML, combining multimodal bioprinting with macrovessel perfusion technology to offer an innovative solution for muscle tissue loss. This promising approach not only represents a breakthrough in tissue repair but also lays the foundation for the future development of more complex tissues and organ printing.