📄 bioengineering

Modular biofabrication of a vascularized skeletal muscle model through endothelialized microvascular seeds

This study presents a modular biofabrication strategy that overcomes vascularization challenges in engineered skeletal muscle by independently maturing and subsequently assembling aligned contractile myofibers with pre-endothelialized microvascular seeds to create a functional, hierarchically organized tissue model.

Original authors: Maiullari, F., Volpi, M., Celikkin, N., Tirelli, M. C., Nalin, F., Viswanath, A., Kasprzycki, P., Karnowski, K., Presutti, D., ?wi?szkowski, W., Costantini, M.

Published 2026-04-01

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CC BY 4.0

Original authors: Maiullari, F., Volpi, M., Celikkin, N., Tirelli, M. C., Nalin, F., Viswanath, A., Kasprzycki, P., Karnowski, K., Presutti, D., ?wi?szkowski, W., Costantini, M.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine you are trying to build a life-sized, working model of a human muscle to help heal a severe injury. The problem is that muscles are hungry; they need a constant supply of food and oxygen to survive. If you just build a block of muscle tissue, the cells in the middle will starve and die because food can't reach them fast enough. You need to build a "plumbing system" (blood vessels) inside the muscle at the same time.

But here's the catch: Muscle cells and blood vessel cells are like two different species that speak different languages. They need different food, different temperatures, and different environments to grow up healthy. If you mix them together in a single pot from the start, they often fight, get confused, or fail to mature properly.

This paper describes a clever new way to solve this problem using a "Modular Construction" approach. Instead of trying to build the muscle and the pipes at the same time, the researchers built them separately in their own perfect environments, and then snapped them together like Lego bricks.

Here is how they did it, broken down into simple steps:

1. The Muscle Factory: Spinning "Spaghetti"

First, they focused on the muscle. They used a special machine called a Rotary Wet-Spinning (RoWS) printer.

The Analogy: Imagine a machine that spins wet spaghetti. It pushes a liquid mixture of muscle cells out of a double-nozzle. The outer layer hardens instantly to form a protective shell, while the inner core stays soft and full of cells.
The Result: As the machine spins a drum, it lays down thousands of these tiny, parallel fibers. Because they are laid down in a straight line, the muscle cells inside naturally line up, just like real muscle fibers. This allows them to grow strong, long, and contractile (able to twitch and move).

2. The Pipe Factory: Growing "Micro-Seeds"

Next, they focused on the blood vessels. They couldn't just print a hollow tube because it would collapse. Instead, they made "Microvascular Seeds" (µVS).

The Analogy: Think of these as tiny, hollow marbles made of a gelatin-like material. Before putting them in the final muscle, they coated the outside of these marbles with blood vessel cells (endothelial cells).
The Magic: They then mixed these coated marbles into a liquid and spun them into long tubes using the same spinning machine. Inside the tube, the marbles acted as scaffolding. The blood vessel cells grew over the marbles and connected with each other, forming a continuous, hollow tube with a clear "lumen" (the hole in the middle where blood would flow).

3. The Grand Assembly: Snapping the Lego Bricks Together

This is the most important part.

The Problem: Usually, scientists try to grow muscle and blood vessels together in one dish. But muscle cells need one type of food, and blood vessel cells need another. It's like trying to feed a cat and a dog the exact same meal; one of them will get sick.
The Solution: The researchers let the muscle fibers grow for 18 days in their perfect "muscle food." They let the blood vessel tubes grow for 12 days in their perfect "vessel food."
The Assembly: Once both parts were fully grown and mature, they took the muscle bundle and the vessel bundle and placed them side-by-side. They used a tiny bit of "glue" (a special gel) to hold them together and let them grow in a shared environment.

Why This is a Big Deal

No Starvation: Because they built the blood vessels inside the muscle structure from the start, the new muscle doesn't have to wait for the body to grow new blood vessels into it. It comes pre-plumbed.
Better Quality: By growing them separately first, both the muscle and the vessels became stronger and more mature than if they had been forced to grow together from day one.
Realistic Size: The tubes they made are about the size of real human arterioles (100–200 micrometers), which is big enough to actually carry blood, unlike many other lab models that only make tiny, useless capillaries.

The Bottom Line

Think of this research as a new way to build a city. Instead of trying to build the houses and the water pipes at the exact same time (which usually leads to a mess), they built the houses first, then built the water pipes, and finally connected them.

This creates a "ready-to-go" muscle model that is strong, organized, and has its own plumbing. This is a huge step forward for treating people with massive muscle loss (like from accidents or burns), as it could eventually lead to transplantable muscle tissues that survive and function immediately after surgery.

1. Problem Statement

The clinical translation of engineered skeletal muscle (eSM) for treating Volumetric Muscle Loss (VML) is currently hindered by the inability to create functional, pre-vascularized constructs that can survive implantation.

Metabolic Demand: Skeletal muscle has high metabolic requirements; without immediate vascularization, constructs larger than the diffusion limit (approx. 200 µm) suffer from ischemia and necrosis before host vessels can infiltrate.
Limitations of Current Models:
- Co-encapsulation: Simultaneously culturing muscle and endothelial cells often fails because the two cell types require conflicting biochemical cues, matrix properties, and differentiation timelines, leading to poor maturation of both compartments.
- Vascular Architecture: Existing models often produce disorganized capillary networks (<10 µm) that lack the structural robustness and physiological diameter (100–200 µm) required for perfusion and integration with host vasculature.
- Anisotropy: Reproducing the aligned, hierarchical structure of native muscle fibers running parallel to vessels remains a significant challenge.

2. Methodology

The authors propose a modular, bottom-up biofabrication strategy using Rotary Wet-Spinning (RoWS) to independently mature muscle and vascular compartments before assembling them into a unified construct.

A. Platform: Rotary Wet-Spinning (RoWS)

A custom 3D bioprinter with a microfluidic co-axial nozzle extrudes core-shell fibers into a crosslinking bath (CaCl₂).
Fibers are collected on a rotating drum (60 rpm) to induce anisotropic alignment.
Core: Fibrinogen-based bioink (biocompatible, cell-supportive).
Shell: Alginate-based hydrogel (mechanically stable, provides structural containment).

B. Modular Component 1: Engineered Skeletal Muscle (eSM)

Bioink: C2C12 murine myoblasts encapsulated in fibrinogen (core) with an alginate shell.
Process: Extruded and cultured in muscle-specific media (differentiation media with horse serum).
Outcome: Formation of aligned, multinucleated myotubes expressing Myosin Heavy Chain (MHC) and sarcomeric $\alpha$ -actinin.

C. Modular Component 2: Microvascular Seeds ( $\mu$ VS)

Fabrication: A "millipede" microfluidic chip generates monodisperse GelMA beads (~110 µm diameter) via step emulsification.
Endothelialization: Human Umbilical Vein Endothelial Cells (HUVECs) are seeded onto the GelMA beads to form a pre-endothelialized "seed."
Vascular Fiber Formation: These pre-endothelialized beads are mixed with single HUVECs and fibrinogen to create a pro-angiogenic core bioink.
Process: Spun via RoWS and cultured for 12 days. The beads act as geometric anchors, guiding endothelial cells to self-organize into continuous, lumenized tubular structures.

D. Modular Assembly

Integration: Mature muscle fibers (Day 18) and vascular bundles (Day 12) are manually co-positioned.
Stabilization: The interface is embedded in GelMA and photopolymerized.
Co-culture: The unified construct is maintained in a shared medium for 7 days to test compatibility and structural stability.

3. Key Contributions

Decoupled Maturation Strategy: Successfully separated the myogenic and angiogenic differentiation processes, allowing each compartment to mature under optimal conditions before integration, overcoming medium incompatibility issues.
Microvascular Seeds ( $\mu$ VS): Introduced a novel modular unit (pre-endothelialized GelMA beads) that guides the formation of physiologically relevant lumens (100–200 µm), overcoming the size limitations of spontaneous capillary sprouting.
Hierarchical Biomimicry: Created a construct that recapitulates the native architecture of skeletal muscle, featuring aligned myofibers running parallel to tubular microvessels within a core-shell fiber bundle.
Scalability: The RoWS platform allows for the continuous, reproducible generation of decimeter-scale constructs with tunable dimensions.

4. Key Results

Muscle Maturation:
- Structure: Myotubes achieved high anisotropy and alignment. Sarcomere organization (Z-disk spacing ~2 µm) was established by Day 10.
- Function: Constructs exhibited spontaneous, coordinated contractions (frequency ~3.1 Hz) and expressed adult myosin isoforms (Myh1, Myh2, Myh4, Myh7) and Titin.
- Mechanics: The alginate shell provided mechanical stability (modulus ~~12 kPa), while the fibrinogen core remained compliant (~~2.3 kPa).
Vascular Maturation:
- Lumen Formation: $\mu$ VS guided the formation of continuous, hollow lumens with diameters of 100–200 µm, matching physiological arterioles/venules.
- Endothelial Phenotype: Cells expressed CD31 and von Willebrand Factor (vWF). Gene expression analysis showed a transition from an activated state (early KDR/VEGFR2 peak) to a stabilized, quiescent phenotype (Day 12).
- Stability: The vascular structures remained patent and did not collapse during culture.
Integrated Construct:
- Viability: Post-assembly co-culture (7 days) showed >80% cell viability in both compartments.
- Preservation: The muscle retained its contractile phenotype, and the vasculature maintained its lumenized structure.
- Crosstalk: Gene expression analysis revealed subtle, compartment-specific adjustments (e.g., mild upregulation of vWF in endothelium) without major transcriptional reprogramming, indicating successful integration without detrimental interference.
- Imaging: Optical Coherence Tomography (OCT) confirmed the 3D spatial organization, showing distinct, parallel muscle and vascular channels within the shell.

5. Significance

Translational Potential: This approach addresses the critical bottleneck of vascularization in tissue engineering, offering a scalable solution for Volumetric Muscle Loss (VML) repair where rapid perfusion is essential for graft survival.
In Vitro Modeling: Provides a sophisticated, physiologically relevant 3D platform to study muscle-endothelial crosstalk, drug screening, and disease modeling, overcoming the limitations of 2D cultures and disorganized 3D models.
Modularity: The concept of "pre-vascularized seeds" ( $\mu$ VS) is versatile; these modular units could potentially be integrated into other biofabrication platforms beyond wet-spinning, enhancing the vascularization capabilities of various engineered tissues.
Future Outlook: While GelMA beads currently persist in vitro, their known degradation profile (2–4 weeks in vivo) suggests they will remodel effectively post-implantation, facilitating integration with the host vasculature.

In summary, this study presents a robust, modular biofabrication pipeline that successfully generates a vascularized, anisotropic skeletal muscle model with functional contractility and perfusable microvessels, bridging the gap between current in vitro models and clinically viable tissue grafts.