Aalto University Develops a Novel Bioink for Cardiac Tissue Applications

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Finland is one of Europe’s most forested nations. Over 70 percent of the country’s boreal forest is covered with spruce, pine, downy birch, and silver birch. But beyond the splendor of the Finnish woodlands, all these trees have one thing in common, and that is nanocellulose. A light solid substance obtained from plant matter which comprises cellulose nanofibrils (CNF) and is considered a pseudo-plastic that possesses the property of specific kinds of gels that are generally thick in normal conditions. Overall, it is a very environmentally friendly and non-toxic substance that is compatible with the human body and has the potential to be used for a range of medical applications.

In 2018, the Department of Bioproducts and Biosystems at Aalto University, located just outside Helsinki, began searching for new ideas to revitalize one of the country’s traditional economic engines, forests (which are handled sustainably thanks to renewable forest resources). At the time, they noticed that one of the possible applications could be working with nanocellulose. Forward two years and the researchers have come up with a new bioink formulation praising nanocellulose at its basis.

Thanks to the structural similarity to extracellular matrices and excellent biocompatibility of supporting crucial cellular activities, nanocellulose-based bioprinting has clearly emerged for its potential in tissue engineering and regenerative medicine. The qualities of the generally thick and fluid light substance make it an excellent match to develop bioinks that are both suitable and scalable in their production, but also have consistent properties. However, there have been major challenges in processing nanocellulose.

As described by Aalto University researchers in a recently published paper in the science journal ACS Publication, the unresolved challenges of bioink formulations based on nanocelluloses are what stops the substance from becoming one of the preferred components for 3D bioprinting structures. This is why Finnish researchers focused on developing a single-component bioink that could be used to create scaffolds with potential applications in cardiac biomedical devices, while fundamentally dealing with some of the limitations of using nanocellulose-based bioinks.

A co-author of the paper and a doctoral candidate at Aalto’s Department of Bioproducts and Biosystems, Rubina Ajdary, told 3DPrint.com that “other than natural abundance and as a renewable resource, nanocellulose has demonstrated to have an outstanding performance in tissue engineering.” She also suggested that “recent efforts usually consider the use of nanocellulose in combination with other biopolymers, for example, in multicomponent ink formulations or to encapsulate nanoparticles. But we were interested in investigating the potential of monocomponent nanocellulose 3D printed scaffolds that did not require crosslinking to develop the strength or solidity.”

In fact, the Biobased Colloids and Materials (BiCMat) research group at Aalto University, led by Orlando Rojas, proposed heterogeneous acetylation of wood fibers to ease their deconstruction into acetylated nanocellulose (AceCNF). As a unique biomaterial opportunity in 3D scaffold applications, the team considered using nanocelluloses due to the natural, easy to sterilize, and high stability porosity of the substance, and chose to introduce AceCNF for the generation of 3D printed scaffolds for implantation in the human body. The team then went on to evaluate the interactions of the scaffolds with cardiac myoblast cells.
“Most modifications make the hydrogels susceptible to dimensional instability after 3D printing, for instance, upon drying or wetting. This is exacerbated if the inks are highly diluted, which is typical of nanocellulose suspensions, forming gels at low concentrations,” went on Ajdary. “This instability is one of the main reasons why nanocellulose is mainly combined with other compounds. Instead, in this research, we propose heterogeneous acetylation of wood fibers to ease their deconstruction into acetylated nanocellulose for direct ink writing. A higher surface charge of acetylated nanocellulose, compared to native nanocellulose, reduces aggregation and favors the retention of the structure after extrusion even in significantly less concentration.”
This is exactly why it was important for the researches to develop a single component bioink. Nanocellulose has shown promises when combined with other biopolymers and particles. However, Ajdary insists that benefits including similarity to the extracellular matrix, high porosity, high swelling capacity, ease of surface modification, and shear thinning behavior of cellulose, encouraged them to study the potential of monocomponent surface-modified nanocelluloses.

Acetylated nanocellulose (Credit: Aalto University School of Chemical Engineering)

The team at Aalto University used the sustainable and widely available nanocelluloses to make several formulations of bioinks and evaluate them, including unmodified nanocellulose CNF, Acetylated CNF (AceCNF), and TEMPO-oxidized CNF.
To 3D bioprint the hydrogels, researchers used Cellink bioprinters, something Ajdary attributed to the user-friendliness of the device and because it provided a lot of flexibility to test different types of hydrogels and emulsions produced in the research group.
In this new process, the single-component nanocellulose inks were first 3D printed into scaffolds using Cellink’s BIO X bioprinter, which is equipped with a pneumatic print head was used to extrude single filaments and form the 3D structures. Then freeze-dried to avoid extensive shrinkage, and sterilized under UV light. After sterilization the scaffold was ready and cells seeded on the samples.
“3D structures of acetylated nanocellulose are highly stable after extrusion in far less concentrations. The lower concentration in wet condition facilitates the scaffold with higher porosity after dehydration which can improve the cell penetration in the structure and assist in nutrient transport to the cells as well as in the transport of metabolic waste,” specified Ajdary.
The researchers claim that the method was successful as the 3D printed scaffolds were compatible with the cardiomyoblast cells, enabling their proliferation and attachment, and revealing that the constructs are not toxic. Although still in research stages, these bioinks and technique can be used for the inexpensive, consistent fabrication and storage of constructs that can be applied as base materials for cardiac regeneration.
What is novel in this study is the particular focus on single-component nanocellulose-based bioinks that open up a possibility for the reliable and scale-up fabrication of scaffolds appropriate for studies on cellular processes and for tissue engineering. Since this is an ongoing research, we can expect to read more published material from Aalto University researchers as they continue testing their unique technique even further.

Scaffolds corresponding to 3D printed AceCNF (Credit: Aalto University)

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