December 1, 2016 (Vol. 36, No. 21)

The Creation of 3D Mosaics Is an Art and a Science

Materials conserved. Assembly procedures streamlined. Time and money saved. All this is possible with “additive manufacturing,” the fabrication of three-dimensional (3D) objects by depositing, one by one, ultrathin layers of material.

The stratum-by-stratum strategy is not only a way to add bulk, it is also, in aggregate, a way to realize immense economic and environmental benefits.

Additive manufacturing is spurring innovation across industries. For example, in automobile manufacturing, General Motors is utilizing 3D printing for rapid prototyping. The company is additively manufacturing ~20,000 unique parts per year. In aviation, General Electric is printing complex parts for aircraft engines. And biotech companies are developing 3D printing applications as well. They are showing marked progress in customized medical products and tissue engineering.

Biotech applications of digital manufacturing also enjoy political support. The Obama administration has launched several public-private initiatives. For example, the “America Makes” Institute, founded in 2012, and the Advanced Tissue Biofabrication Manufacturing Innovation Institute (ATB-MII), announced last June, are government-sponsored efforts to bridge the gap between basic research and commercial manufacturing for 3D printing and novel manufacturing technologies. These collaborations are expediting the bench-to-bedside deployment of additive manufacturing.

As challenges unique to the field are overcome, it appears inevitable that the 3D printing revolution will endure for a variety of biotechnology applications.

Upgrading Microfluidics

Traditionally, the microfluidics field relied on micromolding, a precision injection molding technique, to manufacture medical devices for both R&D and clinical applications. While micromolding is capable of unsurpassed resolution, it is not preeminent in every respect. For example, it looks to fall behind 3D printing as a means of rapid prototyping. Because microfluidics developers appreciate the advantages of rapid prototyping—shorter timelines and trimmer budgets—they are working to bridge 3D printing design and micromolding manufacturing.

Albert Folch, Ph.D., associate professor at the University of Washington, describes the enormous economic incentive that is driving these worlds together: “3D printing provides the flexibility to design extremely complex structures at low cost: complexity, variety, and modeling are essentially free. We can understand how it will work before we make it. There is a huge efficiency in design there.”

Dr. Folch envisions microfluidic devices that will be as “easy and intuitive to use as smartphones.”

Affordable, accessible microfluidics have substantial implications. Dr. Folch is working to bring what he calls “functionalized chemotherapy,” a type of personalized, microfluidics medicine, to glioblastoma patients. Currently, only 30% of patients who have undergone surgery to remove a tumor respond to subsequent chemotherapy. A better response rate could be achieved, however, if chemotherapy selection were to be individualized.

Dr. Folch proposes that a slice of brain tissue from each patient should be kept alive and exposed to different drug combinations in a microfluidic chamber while the patient recovers from surgery. Then, by evaluating the effects on a patient’s brain tissue, scientists may be able to determine which treatments will elicit a response from that particular patient in advance. This noninvasive analysis, performed on tissue outside the patient, could increase cancer treatment effectiveness in an individualized way.

But the resolution possible with 3D printing is “not there quite there yet,” states Dr. Folch. Photolithographic molding can achieve precision of ~1 μm and thus remains the gold standard, outclassing 3D printing. Inadequate resolution, however, is a problem 3D printing is determined to solve. In fact, it seems that manufacturers of 3D printers will soon catch up to their injection molding counterparts.

The other issue that is holding back the field right now is resin quality. “Right now, cells will die in a petri dish that is 3D printed,” explains Dr. Folch. His laboratory is working on this limitation, testing biocompatible, open-source polymers. “It is important to develop open-source polymers,” asserts Dr. Folch. “They should be accessible.” His team is having success with polyethelene glycol dyacrylate (PEG-DA), a neutral polymer used in medical implants and drugs.

In this image, a 3D-printed four-valve switch (left) is shown connected to a 3D-printed cell culture chamber (right). Each valve is pneumatically operated through one of four dedicated tubes, each of which contains a different food-coloring dye. Only the blue-dye valve is open. [Anthony Au, Ph.D., and Albert Folch, Ph.D., University of Washington.]

Biodegradable Medical Devices

Another application of 3D bioprinting is the generation of fully biocompatible and degradable medical devices for use as surgical tools and implants. David Kaplan, Ph.D., professor and chair of the department of biomedical engineering at Tufts University, has focused on printing medical devices composed of silk polymer gels and foams that meet these essential requirements.

Dr. Kaplan’s laboratory has printed surgical clips that have several desirable properties: they are 100% degradable in the body over time; they are composed primarily of silk protein and water, making them both biocompatible and multifunctional; and they require no additives to induce stability via crosslinking.

He admits that more research is needed, especially in creating versatile bioinks and maintaining cells, but Dr. Kaplan remains optimistic: “There is every reason to think that 3D bioprinting will become a staple for therapeutic applications. Imagine an orthopedic surgeon sending an image to the bioprinter in much the same way that a doctor calls in a prescription to the pharmacist now. The individualized prosthetic will be printed by preprogrammed specifications from fully biocompatible, degradable material, and the patient will subsequently receive a completely personalized implant. A perfect fit.”

This sort of application is not just distant speculation. Dr. Kaplan has already had success printing cheeks derived from silk-based bioink to match the concave facial structure of patients in need of an implant.

Vascularization and Scalability of Human Organs

Probably the most touted potential that bioprinting conjurs is that of personalized, on-demand printed tissues for human transplant. For more than 10 years, the field has been in pursuit of this goal, and researchers are making substantial progress, particularly  with respect to bioprinted cartilage. But cartilage is not a vascularized tissue, and the fabrication of such tissue remains challenging.

Racing to clear this hurdle is Ibrahim Ozbolat, Ph.D., associate professor of engineering science and mechanics and biomedical engineering at Penn State University. He has made progress generating murine pancreatic tissue for use as a drug testing model. Although his model tissue is small, Dr. Ozbolat has achieved optimal vascularization and cell integration.

“Bioprinting, as a proof of concept, has now been adequately addressed with the successes in nonvascularized tissues,” asserts Dr. Ozbolat. He confirms that vascularized tissue remains a challenge, and adds that volume size, too, must be addressed. “To scale printed tissue to be sufficient volume for human transplant is not trivial,” he advises.

The physiologic relevance of the cell types and other “bioink” materials being used is also a primary focus. “We use a gel-free approach as a means of maintaining the most physiologically relevant conditions,” states Dr. Ozbolat.

Another challenge for the field, according to Dr. Ozbolat, is an undersized workforce: “We need to expand education and training in this arena to be competitive. Each tissue or organ being printed requires specific expertise. There is a great push now, both politically and from a technical standpoint, that will drive this manufacturing forward.”

Recapitulating Ultrastructure In Vitro

Although printing human organs for transplant remains an ambition for many researchers, there are also many practical in vitro applications in development. Many researchers, including those affiliated with Organovo, a pioneering 3D bioprinting company, are keeping their eye on long-term goals while accomplishing near-term tasks.

“Our immediate goal is to provide better models in a dish for testing things like drug efficacy and toxicity and for recapitulating human disease,” says Deborah G. Nguyen, Ph.D., senior director of R&D, Organovo.

The exorbitant cost of drug development, due in large part to an unremitting failure to predict translation of results from preclinical development to the clinic, is driving pharmaceutical companies to focus on bioprinted miniature human tissues as models for drug screening. Organovo’s approach is unique in that it is “cell agnostic”—it is applicable to any cell type or tissue of interest.

“The design strategy,” explains Dr. Nguyen, “is to look to Mother Nature as a guide, and to place our bioinks in the most physiological context possible—not on plastic, not in isolation.” From there, Organovo researchers place primary cells, with or without hydrogel, into the bioprinter and efficiently print structurally accurate, compartmentalized human cell–derived tissue along the computer-programmed xy, and z axes. “A 24-well plate of 3D liver tissue models, for example, takes about 30 minutes to print,” notes Dr. Nguyen.

The models may be quick to generate, but in culture they have staying power. “We have optimized the technology such that we can maintain live, bioprinted liver tissue in culture for up to six weeks,” asserts Dr. Nguyen. This allows for longer-term testing of compounds and endpoint assessment at various follow-up stages.

Another big advantage is the ability to visualize damage using traditional histological techniques. “Our liver tissues can show us if a compound will be metabolized into a toxic metabolite, as well as give a sense of structural consequences to real tissue that would be impossible to determine with traditional 2D cultures,” states Dr. Nguyen. The 3D cultures also show sensitivity to known liver toxins such as acetaminophen, indicating that they may serve as sensitive, predictive liver toxicity markers in vitro.

Organovo’s bioprinting process can be tailored to produce tissues in formats that are suitable for different applications. For example, in this image, microscale liver tissues have been produced that sit within standard multiwell tissue culture plates and are ready for drug testing. This technology also has the potential to “print” larger therapeutic tissues suitable for transplantation.

Right Place at the Right Time

The versatility of 3D bioprinting lies in the standardization of the material behind the platform. Enter Cellink, a bioink company marketing adaptable, chemically inert scaffolding material and cost-effective bioprinters for diverse research applications.

At Cellink, CEO and co-founder Erik Gatenholm admits it may be some time before 3D bioprinters gain the ability to produce fully functional, vascularized human tissues ready for transplant. Nonetheless, he is exceedingly optimistic.

“The timing is right at the moment,” he insists. “Political hurdles are being removed, and the opportunity to apply this technology in research is here.”

Along with the European Union’s market ban on all animal testing for cosmetics that was enacted in 2013, comes a market for an alternative biological model. “Skin is our biggest application right now,” says Gatenholm. “It’s external, easy to print, and in-demand.”

The challenges that remain are familiar to any new technology and include “educating the market and setting regulatory guidelines so that companies and researchers can implement 3D bioprinting technologies.” While it remains unclear exactly how the FDA will classify 3D printed tissue at this point, Gatenholm envisions “bioprinting as an ultimate replacement for animal testing.”

Moreover, Gatenholm recognizes the potential for achieving personalized medicine by combining 3D bioprinting of tiny, personalized organs with another cutting-edge biotechnology, the organ-on-a-chip. “Combination of these technologies is where the future of pharmaceutical testing is headed,” predicts Gatenholm.

Researchers at Cellink use additive bioprinting techniques to ensure precision and flexibility for manufacture of living human tissue. In this case, bioink composed of living cells and neutral scaffolding was used to produce living cartilage tissue that sustains the structure of a human nose.

Bioinks in Organ Printing

In 3D bioprinting, enabling technologies are becoming more sophisticated and applications are becoming more varied. Activities now being implemented include the printing of structures providing either tissue and organ models or engineered human tissues (and eventually organs) for regenerative medical applications. Bioinks, the solutions actually deposited during printing, are important to the process.

“They’re typically a mixture of living cells and dozens of low and high molecular weight components which support both the mechanical aspects of the printing, as well as the health of the particular cell types employed,” says Bill Whitford, strategic solutions leader, GE Healthcare.

“The absence of optimized and readily available serum-free bioinks supporting this is of growing concern,” he continues. “Bioinks provide many individual features. They must support the structural integrity of the printed construct through the particular printing technique employed, while maintaining robust cell viability and functional cellular phenotype.”

 According to Whitford, GE Healthcare is examining the need of such bioinks to provide an ambient environment that supports the stable culture of stem cells, co-culture of diverse differentiated cells, vasculogenesis, and many chemistries involved in the structural matrix formation.

Cellular requirements can include optimized primary metabolite ratios, growth/attachment factors, heightened pH buffering capabilities, protection from such printing-stresses as hydrodynamic or dehydration forces, inhibition of apoptosis, high-density and 3D culture support, either promoting or inhibiting post-printing differentiation, accommodating the possibility of heightened leachables and particulates, and tolerance to any polymerization chemistries involved in the matrix formation.

“Printing and structural requirements can include controlling such rheological factors as viscosity and surface tension, optimization of the matrix component, and supporting the supramolecular chemistries of hydrogels, colloids, or self-assembly matrices,” notes Whitford. “Also, the inks must compensate for heightened sorption of lipophilic vitamins or lipids or the reported matrix-sequestration of culture factors.”

For Surgeons, the Chance to Practice on Bioprinted Organs Makes Perfect

“Today, surgeons choose from a catalog of existing implants, and they can even become designers, producing implants from a digital file based on data from patient measurements,” says Frédéric Vacher, director of corporate strategy innovation, Dassault Systèmes. “The next step, combining 3D-printed implants with patient cells, holds great promise for tissue engineering.”

To prepare for this step, Dassault Systèmes has arranged for its 3DExperience Lab to work with BioModex, a start-up specializing in surgical simulation. Founded by Sidarth Radjou and Thomas Marchand, BioModex offers surgeons the opportunity to train on a replica of an organ before an operation. “The replica is produced using 3D printing,” explains Marchand. “It is made from different types of plastic, which are combined to replicate the organs. They react to pressure, incision, and separation, just like living tissue.”

Dassault Systèmes is providing BioModex with access to its FabLab as well as its applications for organ design, enabling the start-up to manufacture its first prototypes.

BioModex uses cutting-edge additive manufacturing techniques that can produce the most complex parts of the human body with precision finer than a millimeter, in a single pass. The young company has even succeeded in producing the smallest joint in the human body, the ossicles in the middle ear! This feat is achieved by the ability to print an infinite color palette and levels of mechanical resistance (soft, hard, etc.) at the same time, without the need for additional processing.

In comparison with a purely digital simulation, BioModex offers an unparalleled level of similarity with the physical actions performed during a surgical operation. Surgeons learn and repeat these actions in order to perfect their technique. The physical model, which is created using a series of digital replication procedures, results in a safer surgical operation and offers an entirely new perspective to surgeons.

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