Think about the precision of a 3D printer - now envision it creating complex, finely detailed structures that actively support biological processes and can help regenerate damaged tissues and organs. This is part of the groundbreaking research by scientists from the InterLynk project who are working hard to make such technologies a clinical reality. Recently published in the prestigious journal Small Methods, a team of researchers from the MERLN Institute at Maastricht University, Universidade de Aveiro, and Universidade do Porto, led by Dr. Monize Caiado Decarli and Prof. Lorenzo Moroni, has unveiled a transformative supporting bath for bioprinting called CLADDING.

The Challenge of Printing with Bioinks

Bioinks, the “ink” used in bioprinting, are typically made from biological materials like proteins and other biomolecules, although they can also include synthetic components and living cells. They have enormous potential for creating complex tissue structures that can replace damaged tissues. However, most bioinks suffer from a critical flaw – they are too fluid. Low viscosity means they cannot maintain their shape during the printing process. This is where the CLADDING technique shines, addressing the core problem with a novel solution.

Introducing CLADDING: Crystal Self-Healing Embedding Bioprinting

The CLADDING technique, short for Crystal Self-Healing Embedding Bioprinting, leverages a special support bath to hold the bioink in place during the deposition process. In this process, the bioink is carefully dispensed into the support bath, which physically stabilizes the ink and allows it to maintain its shape. Inspired by a microencapsulation process known as complex coacervation, CLADDING produces a transparent, self-healing bath made of gelatin-gum arabic microparticles inspired by the FRESH method. These particles aggregate to form a colloid with stability at both microscopic and macroscopic levels, preventing the bioink from spreading out and losing shape during printing.

What makes CLADDING truly remarkable is its transparency. The support bath allows light to pass through with much higher efficiency compared to other embedding bioprinting methods, which is essential for the photopolymerization process - a method that uses light to solidify the bioink. This means that even very fluid bioinks can now be printed with high precision, maintaining their shape and structure.

The Science Behind CLADDING

Creating the support bath was no simple task. The researchers meticulously characterized seven gelatins to understand their properties, such as molecular weight distribution, isoelectric point (the pH at which a molecule carries no net charge) and ion content. They found that not all gelatins are created equal—some didn’t meet the necessary criteria for forming a stable support bath.

Through a series of experiments, they optimized the parameters (pH, temperature, hydrophobic character, among others) of gelatin and gum arabic to produce microparticles that form the ideal support bath. This bath not only holds the bioink in place but also self-heals immediately, meaning it can recover its structure after being disrupted, much like a sponge that regains its shape after being squeezed.

Real-World Applications

One of the most exciting aspects of CLADDING is its use of innovative bioinks derived from human blood components. The research team successfully bioprinted constructs using methacrylated platelet lysates (PLMA) and serum albumin hydrogels. These are special mixtures made from human blood proteins that can form stable gels when exposed to light, a process known as photopolymerization.

To make these bioinks even more powerful, the researchers combined them with two types of human cells: human mesenchymal stromal cells (hMSCs) and human umbilical vein endothelial cells (hUVECs).

Human Mesenchymal Stromal Cells (hMSCs): These cells are a type of stem cell that can develop into different types of tissues, such as bone, cartilage, and fat. They play a crucial role in repairing and regenerating damaged tissues.

Human Umbilical Vein Endothelial Cells (hUVECs): These cells line the interior surface of blood vessels and are essential for forming new blood vessels (angiogenesis). This is important for ensuring that any bioprinted tissue can develop its own blood supply to sustain itself.

By incorporating these cells into the bioink, the researchers created bioprinted constructs that not only had the structural integrity needed for tissue engineering but also exhibited excellent cell viability. This means that the cells remained alive and functional within the printed structures, forming dense networks essential for effective tissue regeneration. The constructs maintained their architecture and stability for over a month, demonstrating their potential for long-term applications in tissue engineering.

A Leap Forward in Tissue Engineering and Regeneration

The CLADDING technique represents a substantial leap forward in the field of biofabrication. By enabling the bioprinting of low-viscosity highly biocompatible bioinks containing globular proteins and no additional thickeners, it opens up new avenues for tissue engineering and regeneration. The ability to print complex, stable structures with human-derived materials could pave the way for advanced regenerative therapies.

Reference:

Caiado Decarli, H. P. Ferreira, R. Sobreiro-Almeida, F. C. Teixeira, T. R. Correia, J. Babilotte, J. Olijve, C. A. Custódio, I. C. Gonçalves, C. Mota, J. F. Mano, L. Moroni, Embedding Bioprinting of Low Viscous, Photopolymerizable Blood-Based Bioinks in a Crystal Self-Healing Transparent Supporting Bath. Small Methods 2024, 2400857. https://doi.org/10.1002/smtd.202400857