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Use of Simulation to Evaluate How Well 3D Printing Bioinks Work

[Image: CollPlant]

Plenty of research has been completed regarding the different materials we use to create biomedical parts. Many innovative bioinks – biomaterials loaded with cells to 3D print biological structures – have been developed for 3D bioprinting purposes, from materials like stem cells, gelatin hydrogels, and even sugarcane waste. 3D bioprinting itself is changing the field of medicine as we know it, because we can now fabricate patient-specific human tissues in a laboratory setting.

However, this technology only works if researchers and doctors have good bioinks on hand…and how do we know the materials are good? It’s expensive, difficult, and can take a long time to evaluate if these bioinks are 3D printable. That’s why many many researchers, like a team from the Wallenberg Wood Science Center (WWSC) in Sweden are starting to rely more and more on computer simulations to optimize these biomaterials.

Kajsa Markstedt, a PhD student of chemistry and chemical engineering and biopolymer technology at WWSC, and her colleagues recently partnered up with Johan Göhl’s Computational Engineering and Design team at the Fraunhofer Chalmers Centre (FCC) to test out a process for using a computational fluid dynamics tool to model the way bioinks are dispensed.

“As well as allowing us to evaluate the printability of a bioink, simulations could also help us choose the printing technique that should be employed depending on the target tissue. Such techniques vary depending on the viscosity and nature of the ink being printed, and include ink-jet printing, laser-induced forward transfer, microvalve- and extrusion-based bioprinting,” said Markstedt.

“To model how a bioink is dispensed, we used its mass flow rate and density as input in our calculations. These parameters are the ones most commonly evaluated in experiments when printing designs such as lines, grids or cylinders.”

The team published a paper, titled “Simulations of 3D bioprinting: predicting bioprintability of nanofibrillar inks,” in the Biofabrication journal; co-authors include Göhl, Markstedt, Andreas Mark, Karl Håkansson, Paul Gatenholm, and Fredrik Edelvik.

The abstract reads, “To fulfill the multiple requirements of a bioink, a wide range of materials and bioink composition are being developed and evaluated with regard to cell viability, mechanical performance and printability. It is essential that the printability and printing fidelity is not neglected since failure in printing the targeted architecture may be catastrophic for the survival of the cells and consequently the function of the printed tissue. However, experimental evaluation of bioinks printability is time-consuming and must be kept at a minimum, especially when 3D bioprinting with cells that are valuable and costly. This paper demonstrates how experimental evaluation could be complemented with computer based simulations to evaluate newly developed bioinks. Here, a computational fluid dynamics simulation tool was used to study the influence of different printing parameters and evaluate the predictability of the printing process. Based on data from oscillation frequency measurements of the evaluated bioinks, a full stress rheology model was used, where the viscoelastic behaviour of the material was captured.”

Visual comparison between (L) photo of printed grid structure and (R) simulation of printed grid structure when using 4% CNF ink.

According to Markstedt, 3D printability of a bioink is most often determined by the ratio of line width to the diameter of a 3D printer’s nozzle, the curvature of 3D printed lines, and how many layers can be printed before structure collapse. The FCC scientists also used a dynamic contact-angle model, which uses surface tension and a contact angle as input, to the bioinks’ wettability on a substracte.

“In our simulations, we also used the printing path of a grid structure as input,” Markstedt said.

The full rheology model was based on the material’s viscoelastic behavior and the ink-oscillation frequency data obtained in the team’s experiments. For cellulose nanofibril (CNF) bioinks with different rheological properties, simulations produced outcomes that were similar to experimental results in lab evaluations. Additionally, the researchers could use the computer model the follow the real-time 3D printing process and study the behavior of various inks during dispensing.

Markstedt said, “In experimental evaluations, we often only have the properties of the final, printed grid structure to go on. This is a time-consuming way to develop new bioinks or to optimize printing parameters for a specific ink. It is also expensive since the prepared bioink containing cells is precious.”

It’s also important to test the biostructure soon after it’s 3D printed, because the cells are still viable at that point; this limits how long evaluations can last.

“This often leads to many bioinks being printed at printing parameters that have not been optimized for a specific bioink composition. The result is that the right architecture is not produced, which can be catastrophic because the printed tissue does not function properly,” said Markstedt. “For example, the printed line may be too thin causing the structure to break, or too thick, which prevents nutrients and oxygen reaching all the cells in the bioink.”

Comparison of the distribution of viscoelastic stresses in lines printed with 4% CNF ink and ink 6040 at 0.3, 0.4 and $0.5,mathrm{mm}$ distance between nozzle and plate.

The researchers are fairly certain that their new simulation tool will be able to provide them with far more feedback during 3D printing, like how viscoelastic- and shear stresses are distributed in the ink, while still surmounting all of these issues.

Markstedt said, “This provides a better understanding of why certain printer settings and bioinks work better than others. For example, it allows us to isolate individual parameters, such as printing speed, printer nozzle height, ink flow rate and printing path to study how they influence printing.”

The team will now work on modeling bioink flow inside nozzle geometries that are pre-defined.

“This addition to the model will allow us to observe what effect shear stresses from the nozzle have on the printing process. This will help us to determine how different printing pressures and nozzle shapes affect the bioprintability of a bioink,” explained Göhl.

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[Source: Physics World / Images: Göhl et. al.]

 

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Student Uses 3D Printing to Develop Vocal Prosthetic

Nikita Dubrovsky

Cancer of the larynx is treatable, but it sometimes requires removing the larynx altogether, leaving the patient unable to speak. There are, however, vocal prostheses that can be implanted to restore the patient’s speech. Unfortunately, these prostheses are expensive and tend to need frequent replacement. So South Ural State University student Nikita Dubrovsky is working on an alternative.

“Everywhere in the world, the method being chosen for vocal rehabilitation of the patients who underwent laryngectomy is the voice prosthetic care,” said Dubrovsky. “Most often abroad-manufactured apparatus are used which allow patients to recover their voice. But the big minus here is that such an apparatus is due for replacement after approximately just one year. Imported prostheses are quite expensive, so we decided to come up with our own development, which will feature similar technical characteristics, but will be much cheaper.”

Vocal prostheses first began being used in 1980. A shunt is placed between the trachea and upper esophagus, and a valve is implanted into the opening. This valve allows for the exhalation of air from the trachea to the esophagus, which creates vibration in the walls of the esophagus and the lower pharynx, generating sound. These valves have more than one drawback, however.

“The abroad-manufactured prostheses mostly use plastic, which is very inconvenient because it’s hard,” explained Dubrovsky. “We’re planning on using food silicones, which will make the prosthesis softer, and patients will less suffer from pain. The forms for drip moulding, which we will be filling with silicone, will be manufactured using 3D printing.”

A 3D model of the vocal prosthetic was created, and scientists at the SUSU Research Center for Sport Science have used SLA 3D printing to create a prototype, which will be sent for clinical testing at the Chelyabinsk Regional Center for Oncology and Nuclear Medicine.

“Unfortunately, when we were working with 3D printing, we faced damages that occurred in the drip-moulding tank in the process of the model creation,” said Dubrovsky. “Moreover, such process of production may take 12 to 24 hours, which is a very long time. Since 3D printing turned to be not that accurate, we will probably have to give it up in the future and turn to lathe operators instead, as they are in command of high-accuracy equipment. But first we need to improve our computer model.”

Dubrovsky and his fellow researchers will continue work on the implant as Dubrovsky continues work towards his Master’s degree; he just graduated with a Bachelor’s in Physical Education. They will work on finding food-grade silicone of optimum hardness for patient comfort.

Dubrovsky’s work is potentially good news for the future of patients who require larynx removal; unfortunately, this week also brings some bad news for Italian surgeon Paolo Macchiarini and his patients. Macchiarini published a paper in 2011 in the medical journal The Lancet regarding an artificial windpipe he had created that was coated with the patient’s own stem cells, which would then develop into mature tracheal cells that would not be rejected by the patient’s body. It sounds like a groundbreaking development in bioprinting, but it turns out that the procedure was worse than ineffective.

Macchiarini and colleagues performed the procedure on a total of eight patients, seven of whom died. The surgeons lost track of the eighth. Macchiarini was associated with the Karolinska Institute, which awards the Nobel Prize in medicine every year. In 2014, several surgeons at Karolinska filed a complaint alleging that Macchiarini had downplayed the risks of the procedure, and that it had been carried out on at least one patient who had not been critically ill at the time.

Paolo Macchiarini [Image: Lorenzo Galassi/AP]

Recently, the new President of the Karolinska Institute, Ole Petter Ottersen, requested that The Lancet retract two papers published by Macchiarini, and the journal obliged, as explained in a recent editorial. The Lancet is a prestigious medical journal that only publishes work after extensive peer review, so such a retraction is extremely rare.

“No ethical permit had been obtained for the underlying research,” said Ottersen. “The research was carried out without sufficient support by preclinical data, and the paper presents its data in a way that is unduly positive and uncritical. The clinical findings reported are not supported by source data.”

Macchiarini and his co-authors were found guilty of misconduct, and two members of the Nobel medicine prize assembly were forced to step down.

Discuss these and other 3D printing topics at 3DPrintBoard.com or share your thoughts below. 

[Sources: South Ural State University, CTV News / Images: Viktoria Matveichuk unless otherwise noted]

 

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Indiana University and Lung Biotechnology Partner to Advance 3D Printing of Organs

One of the reasons why I really enjoy writing about 3D printing is because it is a fabulous mix of the fun and the important. The stories we cover can be anything from the enjoyable experience of standing before a giant 3D printed skeleton to the very serious nature of fabricating necessary objects on a 3D printer onboard the International Space Station. The work done by these machines and the people that operate them has, time and again, proven to offer a helping hand (sometimes literally as in the case of prosthetics) to boosting the quality of life of a growing number of individuals.

From left to right: Lester Smith, PhD, Burcin Ekser, MD, PhD and Ping Li, PhD [Image:Eric Schoch, IUSM]

One area in which 3D printing is making particularly important contributions is in the field of medicine. Over the past several years, we have seen stories about students getting hands-on experience through 3D printed models and of improved patient outcomes as a result of preparation and the fabrication of custom surgical equipment for the medical team. With the introduction of bioprinting, the ultimate dream in medicine has been to advance to the point of being able to 3D print whole organs that could be used to replace those that are failing in patients. Thus far, that is still a dream for the future, but important advances are being made in that direction, sometimes great strides, other times only baby steps.

One of the most recent steps forward has come in the form of an agreement between faculty at Indiana University (IU) School of Medicine and the Maryland-based company Lung Biotechnology PBC, that is focused on organ transplantation technologies. The hope is that the $9 million project will result in the knowledge necessary to make the dream of 3D printing organs into a reality. They won’t be starting from scratch; the IU team is already able to generate tissues, but they will use the funding provided through this partnership to analyze the tissues and their structures in order to possibly unlock the key to more advanced organ creation. Dr. Lester Smith, an Assistant Professor of Radiology and Imaging Sciences at IU School of Medicine and the head of the research team, explained the prolonged nature of any such investigation:

“[I]f someone has a skin burn, maybe we can replace skin. Or if someone has a bad liver then we can replace the liver entirely. But this is way down the road. Most of our tissues which make up our organs have a lot of different cell types. They are also vascularized, which means they have a lot of blood vessels that are basically channeling through them. When we get there that’s when I can tell you how long it took. That’s because the body is so complex and there’re so many different parts and so many responses. I couldn’t tell you how long it would take but we’re on the road to that destination.”

Luckily, Indiana University and Lung Biotechnology don’t have to make all the headway by themselves; there are a large number of organizations, from large to small and public to private, pursuing the dream of fabricating organs. This is more than just an effort to do something to see if it can be done; there are people dying every year because they cannot get access to the organs that they need, and further deaths and astronomical medical expenses to deal with for those whose bodies strongly reject the foreign organs. Should it become possible to create a custom organ for someone using their own cells, the entire process from the surgery to simple day to day functionality would be vastly improved, and this partnership should help push that research closer to the gold standard.

What do you think of this news? Let us know your thoughts; join the discussion of this and other 3D printing topics at 3DPrintBoard.com or share your thoughts below.

[Source: Indiana Public Media]

 

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Zero Gravity Bioprinter is Ready for Delivery to the International Space Station

An amazing thing happened two years ago, when Techshot and nScrypt worked together to 3D print a human heart – in zero gravity. Not a working heart, yet, but the two companies were the first to 3D print cardiac and vascular structures in zero gravity using adult human stem cells. Why would they do such a thing, you might ask? The reasoning is much more than “because they can,” as 3D bioprinting in zero gravity is actually easier and more effective than it is on Earth.

Earth-based bioprinting requires thick, viscous bioinks that can contain chemicals or other materials necessary for providing structural support. The lack of gravity in space, however, means that thinner, purer bioinks can be used, as well as thinner print nozzles, allowing for more precision and control. When the first viable human organ is 3D printed, it may very well happen in space.

After nScrypt and Techshot pulled off their successful zero-G bioprinting feat, they began work on the 3D BioFabrication Facility, or BFF, a 3D bioprinting system for the International Space Station. The BFF can 3D print thick tissue and organs using adult stem cells. The printing will happen on an nScrypt 3D Bio Assembly Tool, or BAT; the bioink will be printed into a specialized cell culturing bioreactor cassette designed by TechShot and conditioned in the TechShot ADvanced Space Experiment Processor (ADSEP). The BFF and ADSEP are scheduled to launch on their way to the ISS in February 2019.

The nScrypt BAT 3D printer features high-precision motion and extreme dispensing control, and will use nScrypt’s patented SmartPump, which has 100 picoliter volumetric control and uses super-fine nozzles, down to 10 microns, to dispense biomaterials. This enables the highly controlled and repeatable placement of bioink, which is necessary for printing the fine details of tissues and organs.

“Especially when dealing with something as important as tissue, it is vital to place the correct amount of material in the correct position every time,” said nScrypt CEO Ken Church. “This is what our machines offer and what has contributed to our success in bioprinting as well as other applications. This is an exciting time for discovery and more importantly a time of impact for those that are seriously seeking solutions to grow thick vascularized tissue, which is the basis for a fully printed organ.”

The first complete print, after the initial test prints, will be a cardiac patch for damaged hearts. Cells will be printed into the bioreactor cassette, and the bioreactor will then provide media perfusion to deliver nutrients and remove toxins from the tissue, keeping it alive while providing electrical and mechanical stimulus to encourage the cells to become beating heart tissue.

Rendering of the BFF in an EXPRESS rack [Image: nScrypt]

The BFF may truly be an astronaut’s BFF; in addition to 3D printing tissue for people on Earth, it can print pharmaceuticals and even food on demand for people on the International Space Station.

“We are very excited to see this project, and all that it can provide, come to life,” said Techshot President and CEO John C. Vellinger. “With the goal of producing everything from organs, to pharmaceuticals, to perhaps even food, the BFF has the ability to improve the lives of people on earth and help enable deep space exploration.”

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3D Printing with Magnets in Microgravity

While methods of 3D bioprinting vary, most of them have one thing in common – they print cells layer by layer into a desired shape, which is then transferred to an incubator where it further grows and develops. Alternative methods exist, however, that involve the manipulation of the cell material by magnetic fields. The cells are then “labeled” with magnetic nanoparticles. But now a Russian research team has developed a new method of bioprinting that neither prints layer by layer nor uses magnetic labeling. This method could lead to the creation of radiation-sensitive biological constructs and the repair of organs and tissues.

The new method, which involves magnetic levitation research in conditions of microgravity, was conducted by the 3D Bioprinting Solutions company in collaboration with other Russian and foreign scientists, including the Joint Institute for High Temperatures of the Russian Academy of Sciences (JIHT RAS).

“During the period from 2010 to 2017, a series of experimental studies were carried out aboard the Russian Orbital Segment of the International Space Station with the Coulomb Crystal experimental device,” said Mikhail Vasiliev, head of the laboratory of dusty plasma diagnostics in JIHT RAS. “The main element of the device is an electromagnet that creates a special inhomogeneous magnetic field in which the structures of the diamagnetic particles (they are magnetized against the direction of the magnetic field) can be formed in the microgravity conditions.”

The research was documented in a paper entitled “Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly,” which you can access here. In the study, the researchers describe how small charged particles behave in the magnetic field of a special shape under microgravity or zero-gravity conditions. They also developed a mathematical model of this process based on the methods of molecular dynamics. These results explain how to obtain homogeneous and extended three-dimensional structures consisting of thousands of the particles.

Conventional methods of magnetic 3D bioprinting had several limitations associated with gravity. There are a couple of ways to reduce the power of gravitational forces, one being to increase the power of the magnets that control the magnetic field. This will, however, require a much more complex bioprinter. Another way is to reduce the gravity, which is the approach used by the scientists from 3D Bioprinting Solutions. The method is called formative three-dimensional biofactory, which creates three-dimensional biological structures immediately from all sides, rather than in layers.

The researchers applied the experimental data and the results of the mathematical modeling obtained by the JIHT RAS scientists in order to control the shape of the structures.

“The results of the Coulomb crystal experiment on the study of the formation of the spatially ordered structures led to the development of a new method for the formative 3-D biofactory of the tissue-like structures based on the programmable self-assembly of the living tissues and organs under the conditions of gravity and microgravity by means of an inhomogeneous magnetic field,” said Vasiliev.

Bioprinters based on this new technology will be able to create biological constructs that can be used for many purposes, including estimating the adverse effects of space radiation on the health of astronauts on long-term space missions. It should also be able to restore the function of damaged tissues and organs.

Authors of the paper include Vladislav A. Parfenov, Elizaveta V. Koudan, Elena A. Bulanova, Pavel A. Karelkin, Frederico DAS Pereira, Nikita E. Norkin, Alisa D. Knyazeva, Anna A. Gryadunova, Oleg F. Petrov, Mikhail M. Vasiliev, Maxim I. Myasnikov, Valery P. Chernikov, Vladimir A. Kasyanov, Artem Yu Marchenkov, Kenn Brakke, Yusef D. Khesuani, Utkan Demirci, and Vladimir A. Mironov.

Discuss this and other 3D printing topics at 3DPrintBoard.com or share your thoughts below. 

[Source: Phys.org]

 

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Researchers 3D Print Tissue That Mimics Human Bile Duct

[Image via Cancer.org]

A bile duct plays a crucial role in the body, carrying bile from the liver to the intestine to facilitate digestion. Cancer of the bile duct has an alarmingly low survival rate, and treatment requires that the disease be caught early enough for the affected part of the bile duct to be removed. But there’s some good news for those suffering from conditions of the bile duct, as researchers at Northwestern University have 3D printed a mini-tissue that mimics it.

The research is documented in a study entitled “Tailoring nanostructure and bioactivity of 3D-printable hydrogels with self-assemble peptides amphiphile (PA) for promoting bile duct formation,” which you can access here. Lead author Ming Yan and colleagues 3D printed a nanostructure consisting of peptides amphiphile, or PAs, bioink and bile duct cells, or cholangiocytes.

“3D-printing has expanded our ability to produce reproducible and more complex scaffold architectures for tissue engineering applications,” the abstract states. “In order to enhance the biological response within these 3D-printed scaffolds incorporating nanostructural features and/or specific biological signaling may be an effective means to optimize tissue regeneration. Peptides amphiphiles (PAs) are a versatile supramolecular biomaterial with tailorable nanostructural and biochemical features. PAs are widely used in tissue engineering applications such as angiogenesis, neurogenesis, and bone regeneration. Thus, the addition of PAs is a potential solution that can greatly expand the utility of 3D bioprinting hydrogels in the field of regenerative medicine.”

The PAs and cholangiocytes were mixed with thiolated gelatin at 37°C and 3D printed at 4ºC using an EnvisionTEC 3D-Bioplotter, one of the most-utilized bioprinters on the market. The material retained integrity as the bioinks printed into filaments capable of supporting multi-layered scaffolds. The researchers stabilized the scaffold by cross-linking a derivative of ethylene glycol with calcium ions; scaffold stability was observed in culture for more than a month at a temperature of 37°C.

First author Ming Yan. [Image: Northwestern via Physics World]

The researchers also explored the use of a laminin-derived peptide (Ile-Lys-Val-Ala-Val, IKVAV) and the influence its inclusion in the bioink would have on the bile duct cells. Laminin is a molecule necessary for cell adhesion, and after bioprinting, the bile duct cells remained viable in vitro. Staining revealed the formation of functional bile-cell-based tube structures; when cultured in IKVAV bioink, the structures showed enhanced morphology, forming functional tubular structures.

This is the first time that a bioink-based system supplemented with PAs was used for bile duct tissue engineering. The research shows a lot of promise; the bioprinted bile ducts as well as in vitro systems created with the bioinks have the potential to be valuable for research into bile duct cancer as well as the testing of treatments. Right now, bile duct cancer is a grave diagnosis to receive, but the enhanced research that could be made possible by this work offers hope for better understanding and more effective treatments.

As a next step, the researchers now want to optimize the peptide concentration and test other signaling molecules within the bioinks to enhance the formation of functional tubular structures that mimic those found in the liver.

Additional authors of the research paper include P.L. Lewis and R.N. Shah.

[Source: Physics World]