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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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Researchers Create 3D Printed Bacterial Cellulose Material for Wound Healing

When it comes to medical applications, we’ve seen 3D printing used in the past for healing and repairing wounds, whether through the use of 3D printed bandages, 3D printed blood platelets, or bio-based materials, like nanocellulose. Researchers Dr. Mohamed M. Kanjou, Hassan Abdulhakim, Gabriel Molina de Olyveira, and Pierre Basmaji published a paper, titled “3-D Print Celulose Nanoskin: Future Diabetic Wound Healing,” about using bacterial cellulose for the purposes of wound healing.

“Most 3D printers use heat to melt the plastic or metal to be printed, and biobased materials are degraded,” the team wrote. “But cellulose nanofibrils have a solution to this problem: the printing paste is wet and dries out to a solid material. In this work, it was showed recent wound healing in Vinous Ulcer with kidney and other health complications using bacterial cellulose 3D print membranes.”

[Image: American Process Inc.]

Cellulose nanofibrils, also known as nanocellulose, are made from wood or bacteria, and are the smallest fibers into which cellulose can be decomposed. They can contain up to 50% water, and this viscosity makes it ideal for a 3D printing paste, which can produce strong, biodegradable materials once they’ve dried out. By manipulating the cross-links between the fibrils, the properties can be modified, which allows for the fabrication of strong, porous, and flexible structures.

“Nanocellulose increases the opportunities for creating new materials in wound healing therapy. But this development still requires moisture tests to develops 3D printing with cellulose nanofibrils for medical and biotechnology applications,” the researchers explained.

“Several articles were published by our group since 2015 using Nanoskin membranes for wound healing treatment with successful results in diabetic ulcers, car and other accidents, amputation required ulcers [4] [5] [6]. In this work, it was showed recent wound healing in Vinous Ulcer with kidney and other health complications using bacterial cellulose 3D print.”

Wound healing treated with 3D bacterial cellulose-biological wound dressing (a); developed membrane (b) and Nanoskin developed equipament (c).

This time, the team explored a novel biomaterial and prepared a variety of different bacterial cellulose nanocomposites, such as BC/chondroitin sulfate and hyaluronic acid cross linked with sodium alginate and calcium chloride. They also synthesized bacterial cellulose and bacterial cellulose/chondroitin sulfate/hyaluronic acid.

“The acetic fermentation process was achieved by using glucose as a carbohydrate source,” the researchers explained. “Results of this process were vinegar and a nanobiocellulose biomass. The modifying process was based on the addition of hyaluronic acid and chondroitin sulfate (1% w/w) to the culture medium before bacteria inoculation. Bacterial cellulose (BC) was produced by Gram-negative bacteria Gluconacetobacter xylinus, which could be obtained from the culture medium in the pure 3-D structure, consisting of an ultra fine network of cellulose nanofibers.”

Dr. Kanjou and Abdulhakim supervised the completion of an in vivo analysis – the model was a 60-year-old patient diagnosed with a diabetic foot wound.

Here’s your warning – more icky wound pictures are coming.

Wound healing evolution in 1 month and 3D bacterial cellulose impact use with biological wound dressing.

When the patient, also suffering from kidney failure, arrived at Sheikh Khalifa Hospital, the wound was infected and had accumulated a lot of slough tissue. A classic silver dressing did not show any progress, so the researchers began treating the patient’s wound with 3D printed bacterial cellulose membranes.

For one month, the 3D printed bacterial cellulose material was used on alternating days, to some excellent results – the edge and bottom of the wound were starting to heal, and the wound area was reduced.

Additionally, the slough tissue was easy to remove, and healthy red granulation tissue was starting to grow, which you can see in the below image.

Wound healing evolution in 2 months, 3D print Bacterial cellulose impact use in biological wound dressing.

“Then, after more 1 month, almost all slough tissue is removed by treating with 3-D print Bacterial cellulose only; granulation and building up of healthy tissue is coming up with approximation of skin and the wound is closing,” the researchers wrote.

“Finally, after 4 months of treatment, there is complete healing with minimizing the scar in wound area and able to decrease with time.”

Figure 4. Complete wound healing evolution in 4 months and impact use of biological wound dressing 3D print of bacterial cellulose.

The researchers were able to successfully modify bacterial cellulose by “changing the fermentation medium with hyaluronic acid, chondroitin sulfate, besides of crosslinked with alginate sodium and calcium chloride.” In so doing, they were able to fabricate promising 3D printed scaffolds out of the bio-based material. In addition, the team developed new equipment for carrying out its work.

“In conclusion, 3-D print bacterial cellulose membranes apply to diabetic ulcers, with significant lesions and wound healing requirement; furthermore, natural membranes applications are for all population with different age.”

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REGEMAT 3D Will Start Selling Biomaterials

One of the key players in the bioprinting field in Spain will be incorporating seven new biomaterials. In the coming months, REGEMAT 3D will launch a catalog of biomaterials that customers can buy and use along with their bioprinting systems. According to company officials, in recent years, advances in 3D bioprinting systems have become very important, as well as new biomaterials for regenerative medicine. The performance of the research groups with which they collaborate has produced results that were likely unheard of years ago. Still, they consider that these innovations in bioprinting systems must be accompanied by a progressive definition and characterization of the biomaterials being used. This year, one of REGEMAT 3D’s objective is to advance biomaterials for further research in the different applications derived from the 3D bioprinting sector, which is growing every year.

REGEMAT 3D bioprinting with new biomaterials

Each specific application requires different solutions and appropriate biomaterials to optimize processes. For instance, it is easy to understand that to regenerate skin components, hydrogels, cells and growth factors are different from those needed to regenerate muscle tissue, bone or cornea. So, it is essential to offer researchers and scientists different biomaterials to aid their work. REGEMAT is focusing on seven: thermoplastics, collagens, alginates, agaroses, gelatin methacryloyl (GelMA), nanocellulose, and different types of cell media compatible with the cells used. All of the biomaterials should be easy to print, handle and will allow researchers to tackle some of the challenges they face while working. 

The new biomaterials for 3D bioprinting will be available on the company’s web page (which they will relaunch shortly), as well as through their offices. REGEMAT 3D has agreements with several national and international partners for the manufacture of these products. The first ones to be sold commercially will be nanocellulose, collagen, and alginate.

REGEMAT 3D new biomaterials

The Granada, Spain-based biotech company has sold its machines to users in more than 25 countries. For years, the company has been working with research groups at the University of Granada in advanced therapies, participated in a joint project for the development of new therapies for cartilage regeneration, and has collaborated with the University Hospital of La Paz, where REGEMAT 3D’s founder coordinates the 3D Tissue Engineering and Printing Platform (PITI3D), which provides ingredients and processes to generate functional tissues. Since its origin, the startup has been focusing on regenerative medicine, developing custom hardware and software required and demanded by some of the major hospitals and research universities in the region. They develop their own bioprinting systems – the BIO V1 machines – and customize them for their users’ applications according to the requirements of each investigation.

Last January, a group of researchers led by the University of Granada and REGEMAT 3D, published an academic article on the development of a volume-by-volume 3D biofabrication process that divides the printed part into different volumes and injects the cells after each volume has been printed, once the temperature of the printed thermoplastic fibers has decreased. This helps overcome problems that arise when working in 3D bioprinting with thermoplastics at high temperatures: one of the biomaterials they will soon begin commercializing, with which the company is very familiar and has worked with for a long time. 

To continue developing new biomaterials and launching new products, the Spanish company, led by founder and CEO José Manuel Baena, has managed to raise 320,000 Euros in the midst of the latest financing round. REGEMAT 3D, along with its sister company Breca, are not only launching the new series of biomaterials, but they are also expanding their presence to Brazil, where the company has already started to market its products, and China, where they closed an agreement with Chinese distributor ApgBio, based in Shanghai, that’s responsible for introducing bioprinting equipment in the country for the regeneration of organs or tissues. Companies like REGEMAT 3D are gearing up to mass produce biomaterials, providing researchers with more options when it comes to bioprinting for regenerative medicine and advanced therapies, usually keeping in mind how patients bodies will react to the new materials, and whether they will be compatible. Spain, like many other European countries, is quickly catching up to the world of bioprinting, which today is led by US-based companies but shows promise in many developed countries.

[Images: REGEMAT 3D]

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ORNL and UMaine Initiative Receives Funding to Create New Bio-Based 3D Printing Materials

UMaine Advanced Structures and Composites Center students and staff lift a boat roof from a mold 3D printed with a new biomaterial, nanocellulose-reinforced PLA, developed at the University of Maine. L-R: Michael Hunter, Camerin Seigars, Zane Dustin, Alex Cole, Scott Tomlinson, Richard Fredericks, and Habib Dagher. [Image: UMaine]

The researchers at Oak Ridge National Laboratory (ORNL) in Tennessee have spent a lot of time working with unique 3D printing materials, such as polyester, lignin, and nanocellulose, which is a bio-derived nanomaterial. Now, a new research collaboration between ORNL and the University of Maine’s Advanced Structures and Composites Center aims to increase efforts to use wood products as 3D printing materials. Together, the team will work with the forest products industry to create new bio-based 3D printing materials that can be used to make products like building components, boats and boat hull molds, wind blades, and shelters.

The large-scale initiative was announced this week in Washington, DC. Leaders from the university and ORNL, as well as the DoE‘s assistant secretary for energy efficiency and renewable energy Daniel Simmons, the founding executive director of the Advanced Structures and Composites Center Habib Dagher, and US Sens. Susan Collins, Lamar Alexander, and Angus King were all on hand for the announcement, which also stated that UMaine and ORNL had received $20 million in federal funding for the program from the DOE’s Advanced Manufacturing Office.

[Image: UMaine]

“While Oak Ridge is a global leader in additive manufacturing, the University of Maine is an expert in bio-based composites. By working together, they will strengthen environmentally responsible advanced manufacturing in America as well as helping the forest industry in the state of Maine,” Senator Collins said.

Sens. Collins and King requested federal help to save the declining forest products industry in Maine back in 2016, after several paper mills in the state closed their doors. This led to the founding of the Economic Development Assessment Team (EDAT) to work across agencies in order to come up with economic development strategies for the rural communities in Maine that were suffering from the mill closures. This team resulted in the ongoing partnership between UMaine and ORNL.

“Using Maine forest products for 3D printing is a great way to create new jobs in Maine and a good reminder that national laboratories are our secret weapons in helping the United States stay competitive in the rapidly changing world economy. The partnership between the University of Maine and the Oak Ridge National Laboratory is a model for how science and technology can help Americans prosper in the new economy,” said Senator Alexander.

A 3D printed representation of the state of Maine presented by Habib Dagher, executive director of UMaine’s Advanced Structures and Composites Center. The material was a wood-based product developed at UMaine. [Image: Contributed by the office of Sen. Susan Collins]

This October, ORNL’s BAAM 3D printer will be installed at UMaine, which is actually considered a world leader in cellulose nano fiber (CNF) technology. UMaine students can also visit ORNL’s Manufacturing Demonstration Facility (MDF), while staff from the laboratory can in turn learn about cellulose fiber and composites at UMaine’s composites center.

One of the printer’s first tasks will be to fabricate a boat mold out of a wood-based plastic, though the team hopes to apply the technology to many large-scale manufacturing applications.

Habib Dagher, Executive Director of the Advanced Structures & Composites Center holds up 3D printed representations of Maine and Tennessee signifying new manufacturing programs between UMaine and ORNL that will use wood-based products in 3D printing. Sen. Angus King, I- Maine, and Sen. Susan Collins, R- Maine, watch Dagher’s presentation after announcing $20 million in federal funding for the collaboration. [Image: Contributed by the office of Sen. Susan Collins]

Dagher explained, “The material is nanocellulose, basically a tree ground up to its nano structure. These materials have properties similar to metals. We are taking those and putting them in bioplastics so we can make very strong plastics that we can make almost anything with.”

The team will then add the nanocellulose to PLA.

“The University of Maine is doing cutting-edge research related to bio-feedstocks and the application of advanced manufacturing in regional industries,” said Thomas Zacharia, the director of ORNL. “We are thrilled at this opportunity to expand our research base while providing UMaine with access to the leading national capabilities we have developed at ORNL’s Manufacturing Demonstration Facility.”

The overall goal for the initiative is to find new uses for wood-based products in an effort to reinvigorate Maine’s forest products industry, while also helping to make regional manufacturing stronger by connecting university–industry clusters with the MDF. The federal funding will be divided equally between both facilities.

“We will integrate 20 years of research in bio-based composites at UMaine and 3D printing at ORNL. It is an opportunity engine for our students, faculty, staff and manufacturing industry who will work side by side with researchers at our nation’s foremost research laboratory. Together, we will break down wood to its nanocellulose structure, combine it with bioplastics, and print with it at hundreds of pounds an hour,” said Dagher. “The research we will be conducting with ORNL will spur next-generation manufacturing technologies using recyclable, bio-based, cost-effective materials that will bolster our region’s economy.”

Scientists from UMaine and ORNL will be conducting research in multiple areas, such as multiscale modeling, CNF production, drying, functionalization, and compounding with thermoplastics, and sustainability life-cycle analysis.

CNF could actually rival the properties of steel, and by successfully adding it into plastics, the researchers could create a renewable feedstock for strong, recyclable, bio-derived material systems that might even be 3D printed at deposition rates of hundreds of pounds an hour. Additionally, using a material that’s 50% wood could help open new markets for the forest products industry.

UMaine will get world’s largest 3D printer and use wood-based plastic to make boat molds

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