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Aalto University Develops a Novel Bioink for Cardiac Tissue Applications

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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New Method: Immersion Bioprinting of Tumor Organoids Will Increase the Throughput of 3D Drug Screening

Drug testing and screening for cancer drug discovery can take years and the 2D cell cultures and animal models used to estimate their efficacy before reaching human trials are often not representative of the human body, which is why researchers are turning to bioprinting technologies to increase the success rate during human trials by providing human-specific preclinical data. In 2018 there were 17 million new cases of cancer worldwide, and the disease is expected to affect 27.5 million people each year by 2040, this high incidence level makes tackling the disease enough of a reason for researchers to consider new technologies that could accelerate drug discoveries and screenings. Although still in its lab phase, a new development that uses immersion bioprinting of human organoids could change 3D drug screening.

Researchers from Cornell University, Wake Forest School of Medicine, Virginia Polytechnic Institute and State University and The Ohio State University have published an article in Micromachines, demonstrating an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. Using a hydrogel bioink comprised of hyaluronic acid (HA) and collagen they were able to bioprint it into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form.

According to the article, the use of bioengineered human cell-based organoids may not only increase the probability of success during human trials, but they could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, they suggest that one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high throughput screening, so bioprinting can be used to scale up the deposition of such organoids and tissue constructs.

The team of scientists employed two commercially available bioprinters to evaluate the compatibility of the collagen-HA hydrogel and the HyStem-HP hydrogel: Cellink‘s INKREDIBLE bioprinter and Allevi‘s Allevi2 bioprinter. This method was validated using several cancerous cell lines and then applied to patient-derived glioblastoma (GBM) –a fast-growing brain tumor– and sarcoma (or malignant tumor) biospecimens for drug screening.

For the initial analysis of hydrogel biocompatibility, researchers used two common cell lines: human liver cancer and human colorectal cancer.

While carrying out patient-derived tumor biospecimen processing, they obtained two glioblastomas and one sarcoma biospecimen from three surgically treated patients in adherence to the guidelines of the Wake Forest Baptist Medical Center IRB protocols. These biospecimens were processed into cell suspensions, successfully yielding millions of viable cells from each sample. The cells were then combined with the collagen–HA bioink for deployment in immersion bioprinting. After bioprinting, the GBM and sarcoma patient-derived tumor organoids (PTOs) were maintained for seven days in the incubator, after which a chemotherapy screening study was initiated.

Schematic of the printing process using 2 bioinks in two commercially available bioprinters: Cellink Inkredible and Allevi 2 (Image: Cornell University/Wake Forest)

The researchers claim that while their PTOs have been useful for disease modeling, mechanistic study, and drug development, they have also used these models in a diagnostic sense to influence therapy, which might just be the ultimate goal of their work.

This 3D bioprinting approach called immersion bioprinting is an efficient way to surpass the limitations that have plagued tumor organoid systems. The experts, in this case, suggest that there have been few advances in regard to approaches to the printing process itself, or generation of novel, more user-friendly bioinks. Indicating that “unfortunately, many bioprinting studies are somewhat repetitive, falling back on traditional biomaterials and their crosslinking approaches, which were never developed to be bioprinted or to accurately represent the complexities of the native ECM (extracellular matrix).”

Results of the published study suggests that the realization of this technology that can fabricate PTOs in a consistent and high-throughput fashion will provide a valuable ex vivo/ in vitro tool that can be deployed for many subsequent studies, including target discovery, mechanistic investigation of tumor biology, drug development, and personalized drug screens to aid in treatment selection in the clinic.

Clinical oncology is faced with some critical challenges during this decade, from inefficient trial design to integrating new technologies in diagnostics and drug trails. However, advances in new methodologies, from hardware design to improved bioinks developed specifically for bioprinting, are opening up new opportunities for bioprinting-based applications. This new study, in particular, suggests that with advances in bioprinting hardware, software, functional ECM-derived bioinks, and modifications to printing protocols, bioprinting can be harnessed not only to print larger tissue constructs, but also large numbers of micro-scaled tissue and tumor models for applications such as drug development, diagnostics, and personalized medicine.

Employing bioprinted patient-derived tumor organoids in a clinical precision medicine setting (Image: Cornell University/Wake Forest)

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The Future of Bioprinting Research Has a New Road Map

Improving efficiency, optimizing technology, increasing awareness, even reducing costs and time, these are all traits that result from strategic road maps, and in the case of bioprinting, where the outcomes affect tissue engineering, bespoke outcomes for patients, regenerative therapy, and much more, having a blueprint for the entire industry seems like a bright idea. Especially when this blueprint highlights some of the challenges on the way to achieving meaningful and innovative scientific development.

Published in IOP Science’s Biofabrication Journal, “The Bioprinting Roadmap” features advances in selected applications of bioprinting and highlights the status of current developments and challenges, as well as envisioned advances in science and technology. The roadmap brings together researchers, specialists, and physicians from a myriad of universities, institutions, and hospitals around the world, combining their knowledge to focus on different aspects of bioprinting technology. These include: Jürgen Groll, professor of functional materials in medicine and dentistry at the University of Würzburg in Germany; Binil Starly, professor of mechanical engineering at North Carolina State University; Andrew Daly, an orthopedic surgeon at Emory University Hospital; Jason Burdick, a bioengineer from the University of Pennsylvania; Gregor Skeldon, a life science medical writer at Maverex, in the UK; Wenmiao Shu, professor of biomedical engineering at the University of Strathclyde in Glasgow; Dong-woo Cho, mechanical engineer at Pohang University of Science and Technology; Vladimir A. Mironov, chief scientific officer of 3D Bioprinting Solutions, and many more.

The roadmap focuses on a broad spectrum of topics, in a detailed and readable fashion that showcases broad knowledge of the field by its authors, as well as a great deal of research that went into the making of the guide. The paper is categorized into the following sections:

Charting the progress from cell expansion to 3D cell printing
Examining the developments and challenges in the bioinks used for bioprinting
Looking into bioprinting of stem cells
Presenting a strategy for bioprinting of tissue vascular systems and tissue assembly:
Examining the potential for using 3D printed biohybrid tissues as in vitro biological models for studying disease
Analyzing how 3D bioprinting can be used for the development of organs-on-a-chip
Biomanufacturing of multi-cellular engineered living systems
Exploring how researchers are pushing boundaries with bioprinting in space
Investigating the developments of bioprinting technologies

The introduction of the research work was in charge of Wei Sun, chair professor of the College of Engineering from Drexel University, in Philadelphia, and Tsinghua University in Beijing, China, who said to IOP Publishing that “there are a number of challenges to overcome, including the need for a new generation of novel bioinks with multi-functional properties to better transport, protect and grow cells during and after printing; better printing processes and printers to deliver cells with high survivability and high precision; efficient and effective crosslinking techniques and crosslinkers to maintain the structure integrity and stability after printing; integration with microfluidic devices to provide a long term and a simulated physiological environment to culture printed models.”

Wei Sun (Credit: Drexel University)

“Due to the rapid advancements in bioprinting techniques and their wide-ranging applications, the direction in which the field should advance is still evolving,” went on Sun. “The roadmap aims to address this unmet need by providing a comprehensive summary and recommendations, useful to experienced researchers and newcomers to the field alike.”

The research sheds light on the main roadblocks to overcome in the future. The specialists consider that the next technologies will use “multiple modalities” in one single platform and “novel processes, such as cell aggregate bioprinting techniques” to create scalable, structurally‐stable, and perfusable tissue constructs. But in the meantime the challenges that remain are many. In his introduction, Sun talks about the need for a new generation of novel bioninks with multifunctional properties to better transport, protect, and grow cells during and after printing; better printing processes and printers to deliver cells with high survivability and high precision; efficient and effective crosslinking techniques and crosslinkers to maintain bioink structural integrity and stability after printing, and integration with microfluidic devices to provide a long-term and a simulated physiological environment in which to culture printed models.

Other researchers highlighted the importance of further improving bioreactor-based cell-expansion systems to lower barriers to the adoption of bioprinting in regenerative medicine and tissue engineering product markets. While Skeldon and Shu suggest that stem cell bioprinting can be realized if scientists find a way to reduce the shear stress of bioprinting stem cells.

Another obstruction is the high production costs and the difficulty of having large-scale production of organoids. Jinah Jang and Dong-Woo Cho from the Pohang University of Science and Technology suggest that there have been remarkable advances to the recreation of vasculatures and large organs even though challenges remain immense.

Whereas some of the highlights of the research include novel benefits from bioprinting in space, considering that microgravity conditions enable 3D bioprinting of tissue and organ constructs of more complex geometries with voids, cavities, and tunnels; cell expansion as a critical upstream process step for cell and tissue manufacturing; the great promise of stem cells in biomedical research and applications, which through bioprinting, can be particularly positioned in 3D in relation to other cell types and/or biomaterials, as well as progress in stem cell biology and in vitro culture which is opening up new doors to regenerative medicine and better physiological cell-based assays for disease models.

With so many advances in bioprinting around the world and remaining challenges to overcome, both seasoned researchers and newcomers will find an interesting and complete summary of the bioprinting industry. This game plan can help researchers come together to create new novel processes and fill in current technological gaps, as the researchers suggest.

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