The ability to design three-dimensional (3D) models and prototypes has enabled advancements in many industries. 3D printing, also called additive manufacturing, is the process of creating a 3D model by laying down successive layers of material that build upon each other. When the “ink”—plastics, polymers, metal alloys, or other substances—is infused with living cells, the process is referred to as 3D bioprinting.

Both 3D printing and 3D bioprinting are increasingly being used to advance healthcare. According to a recent report, Global Bioprinting Market: Focus on Product, Application, Technology, Country Analysis, Patent Analysis, Market Share and Competitive Landscape—Analysis and Forecast (2018–2025), the global 3D bioprinting market will reach at least $4.7 billion by 2025.

A significant goal underlying the growth of 3D bioprinting technology is the potential to bridge the enormous gap between the demand and the supply of organs for transplantation. Developments in 3D bioprinting technologies have made possible the printing of relatively simple tissues and structures—sheets of skin or cardiac patches, for example. As the technology matures, more complex organs, such as the retina, liver, and lungs, may follow.

Although researchers are still working to print (and gain FDA approval for) functional human organs, 3D bioprinted tissues already have found numerous applications in drug discovery, testing drug toxicity, tissue engineering, consumer product testing, bone transplants, and cosmetic dentistry.

The advantages of 3D bioprinting, when compared with other tissue fabrication techniques, include fabrication of anatomically correct shapes, fabrication of porous structures, use of multiple cell types, and controlled delivery of growth factors and genes. One of the biggest challenges to overcome includes driving down the resolution of the printing technique enough to enable vascularization of tissues and organs.

The possibilities and challenges of this exciting field were discussed at the SelectBIO: 3D-Bioprinting and Tissue Engineering conference held recently in Boston. A handful of the scientists who spoke at the conference also discussed their research and insights on bioprinting and tissue engineering with GEN.

He Who Controls the Ink…

While a primary focus of the early days of the 3D bioprinting revolution has been advancing printer technology, the thing that the printer spits out—the ink—has received far less attention. That’s where Boston-based company Cellink comes in.

“When I started the company, I realized that there were a lot of printer manufacturers building expensive systems,” said Erik Gatenholm, co-founder and CEO at Cellink. “But nobody had taken the approach of focusing on the inks.”

Gatenholm saw a wide-open opportunity to design and commercialize a suite of standardized biomaterials for bioprinting human tissues and organs.

Cellink’s bioinks consist of variety of materials—including gelatin, collagen, alginate, or other natural polymers—and infused with human cells. The inks are custom-designed for specific tissue types and are compatible with the majority of 3D bioprinters on the market.

When Gatenholm first launched Cellink in 2016, he tried to partner with 3D printing companies or bioprinting companies, but none wanted to collaborate with such an early-stage company. So, Cellink decided to develop its own cost-effective, mobile line of printers. Coupled with the firm’s bioink, Cellink now offers complete 3D bioprinting package solutions for customers, which primarily include academic institutions and pharmaceutical companies. The company has at least 500 systems in use in labs around the world.

Gatenholm said that although Cellink’s bioinks are currently mostly used to create tissue for the purpose of researching and developing new drugs, they have the potential to be used to print replacement tissues for use in humans. While no 3D printed tissue has yet received FDA approval, Gatenholm thinks one of the earliest types to be approved may be cartilage or skin, which are relatively homogenous and should be less complex to manufacture.

In the meantime, Cellink is focused on offering more user friendly yet more advanced systems as well as better bioinks and biomaterials.

3D Printing, but with Lasers

While the prospect of 3D bioprinting new tissues to replace worn out ones has driven much of the interest in the technology, in the near term most of the advances in the field will come from printing miniature copies of different tissues for drug testing and improving our understanding of basic cell biology. And, if Doug Chrisey, Ph.D., professor, Jung Chair of Materials Engineering, Tulane University, has his way, those tiny replicas will imitate the cellular heterogeneity of, and reside in the same microenvironments as, the full-size versions in our bodies.

“Conventional cell cultures often comprise a single cell type,” Dr. Chrisey said. “What we’re trying to do is mimic an entire tissue construct, which is made up of biochemicals, scaffolding, and a great deal of heterogeneous cells that are present in a specific combination.”

Dr. Chrisey and his team have pioneered the use of a technology called laser direct-write (LDW) printing to create structures on the order of a few cubic millimeters on a cell-by-cell basis. This technique gives the researchers the ability to precisely deposit a variety of different cell types with near single-cell spatial resolution.

The team starts by culturing cells that it wants to deposit. Because most cells in the body are adherent, they cut off the cells’ pseudopod binding sites, leaving the cells in a temporary balled-up, nonadherent state. The cells are then loaded into a biopolymer film that is placed above a receiving substrate such as a petri dish or glass slide. A computer then triggers a precise pattern of 248-nanometer excimer laser bursts that produce tiny gas bubbles in the biopolymer. These bubbles propel cells forward and deposit them directly onto the receiving substrate in layers that can be stacked up to form 3D tissues.

Dr. Chrisey and his team have used LDW printing to create many types of complex tissues, including collagen fibers, muscle fibers, and neural circuits. They also found that deposited cells rapidly organize into physiologically functional tissues and that metastatic cancer cells tend to migrate more toward vasculature than non-metastatic cells.

Building Modular Units

Advances in 3D printing are benefitting biology not only through the prospect of printing biological tissues, but also in making it cheaper and more efficient to design tools that biologists use in the lab. One such biologist making the most of modern 3D bioprinting technology is Noah Malmstadt, Ph.D., associate professor of chemical engineering and materials science, biomedical engineering, and chemistry at the University of Southern California.

Dr. Malmstadt and his colleagues use 3D bioprinting techniques to build modular microfluidic systems utilizing standardized parts without having to rely on costly clean room fabrication methods. They primarily use their microfluidics systems to simplify fluid mixing underlying miniature diagnostic tests and high-throughput microbioreactor processes.

Routing fluids through 3D assemblies of microfluidic modules allows for rapid and more precise fluid mixing. One such assembly his research team has designed is a device with a parallel network of four 250-micron-wide tubes. Forcing nonmixing fluids, such as oil and water, through the tubes allows the team to produce an endless stream of nearly identical microdroplets. Each droplet acts as a micro-scale chemical reactor in which chemicals can be mixed with high-levels of precision. The team has used the device to develop a reliable method for producing gold nanoparticles, which have been shown to be an ideal medium for delivering drugs to individual cells. This method has the potential to greatly reduce the cost of generating gold nanoparticles, which currently run $80,000 per gram.

Going forward, Dr. Malmstadt is experimenting with using materials in his printing process that minimize adhesion, and therefore don’t gum up the 150-micron-wide channels he is trying to create. “That’ll really allow us to increase the density of channels on a device and minimize overall device size,” he prediated.

The next big step, though, for Dr. Malmstadt will be integrating off-the-shelf components—including photodiodes, heaters, and sensors—into his devices. “That would really bring our costs down,” Dr. Malmstadt explained. “We’re inspired by the microelectronics industry and their cost-minimization procedures. That’s really what’s driving us and our technology forward.”

Taking 3D Printing to the Next Level

While many researchers are expanding the potential of 3D bioprinting by focusing on characteristics such as spatial resolution and embedded vasculature, others are looking at a more literal expansion of the technology—moving it from three dimensions to four.

4D bioprinting—the printing in 3D of an object with the engineered capability to respond over time to its environment and change in shape (e.g., deforming, twisting, or growing in size) or function (e.g., cellular differentiation, change in cell polarity, or even organ development)—is just one of the many avenues of 3D bioprinting that GE Healthcare is pursuing.

Like many companies involved in the 3D bioprinting field, GE Healthcare also supports the development of novel bioink materials and bioprinting instrumentation. But, according to William Whitford, strategic solutions leader for bioprocesses at GE Healthcare Life Sciences, the healthcare conglomerate is quite interested in peripheral technologies aimed at improving and streamlining the 3D bioprinting process, including imaging analysis and data management tools.

“Imagine you’ve printed a series of organoids and want to see how they respond to certain chemical or biological challenges,” Whitford suggested. “We support the development of high-power microscopes that will let you track those responses in fine detail.”

In addition, GE Healthcare has long been interested in the storage, processing, and transfer of medical images in a digital format. This expertise extends to the digital design models that direct the 3D bioprinting process itself. “Many 3D bioprinted objects are very basic, but they’re becoming much more complicated because we’re moving into multi-material, multi-mode bioprinting that requires sophisticated model construction and control of the printer,” Whitford noted. “GE Healthcare is supporting the 3D bioprinting field in the development of digital modeling and data management tools.”

Treating Rare Liver Diseases with 3D Bioprinted Tissue

Although approximately 17,000 patients in the U.S. are on the waiting list for a liver transplant, only 6000 liver transplants are performed each year. For patients awaiting a liver transplant, a 3D bioprinted liver tissue may help extend survival until a liver is available.

Using its 3D bioprinting technology, Organovo has developed a liver therapeutic tissue, called NovoTissues®, which demonstrated robust function in two models of rare liver diseases, 1) α-1 antitrypsin deficiency (AATD) and 2) hereditary tyrosinemia type 1 (HT-1), says Benjamin Shepherd, Ph.D., senior director, therapeutics, Organovo Holdings.

Organovo’s technology incorporates human liver cells into a printable bio-ink to generate sections of liver tissue by the controlled patterning and deposition of specific cell types. The formed tissues exhibit dense cellularity and develop extensive cell–cell interactions akin to native tissue, resulting in prolonged tissue viability and function outside of the body, according to Dr. Shepherd. These tissues are then implanted into the setting of established animal models of liver disease to test for safety, therapeutic efficacy, and viability.

Organovo’s NovoTissues for the treatment of AATD was granted orphan drug designation by the FDA in 2017. When the bioprinting technique was evaluated in a model of AATD, the implanted tissues rapidly engrafted, demonstrated graft retention, and displayed clear evidence of disease modulation through a 90-day evaluation, notes Dr. Shepherd.

Characterized by a patient’s inability to metabolize the amino acid tyrosine, HT-1 causes severe liver damage, and current treatment options are often limited to organ transplantation. Organovo’s bioprinted liver tissue demonstrated retention and sustained functionality postimplantation in mouse models of this rare inherited disease, notes Dr. Shepherd, adding that the diseased animals showed improvement in liver health and extended survival compared to non-treated animals.

“The success of Organovo’s bioprinted therapeutic tissue in the above preclinical studies highlights its applicability in treating conditions like HT-1 and AATD, where there is critical unmet need for progressive, novel therapies,” says Dr. Shepherd.

Hybrid Additive Manufacturing

Source: 3D Printing and Additive Manufacturing, Vol. 5, No. 3

Producing synthetic scaffolds with adequate physical, chemical, and biological properties remains a challenge for tissue engineering. Internal architecture, surface chemistry, and material properties have strong impact on the cell’s biological behavior.

This requires sophisticated systems not only able to process multiple materials with different characteristics­—creating fully interconnected, 3D porous structures with high reproducibility and accuracy—but also able to modify their properties during the fabrication process.

A study (“Hybrid Additive Manufacturing System for Zonal Plasma-Treated Scaffolds”) by Liu et al. which appeared in 3D Printing and Additive Manufacturing, Vol. 5, No. 3 (published by Mary Ann Liebert, Inc.), introduced a novel additive manufacturing system comprising a multiprinting unit (screw-assisted and pressure-assisted printing heads) together with a plasma unit that enables the surface modification of printed scaffolds. Poly(ε-caprolactone) scaffolds with a lay-down pattern of 0/90° were fabricated using the screw-assisted printing head, and a plasma jet unit was used to uniformly modify each layer (either a specific region of each layer during the printing process or the external surface of the printed scaffolds).

Scaffolds were produced using different plasma exposure times and different distances between the plasma head and the printed layer while using fixed printing conditions. Produced scaffolds were morphologically, mechanically, chemically, and biologically characterized. Results show that the distance between the plasma head and the printed material has no significant effect on the mechanical properties, whereas the increase of the plasma deposition velocity improves the mechanical properties.

As expected, plasma treatment increases hydrophilicity and consequently the biological performance of the scaffolds. Results also show the potential of the proposed fabrication system to create functional gradient or scaffolds with tailored properties.

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