Researchers develop nanoengineered bioink to 3D print functional bone tissue

Scientists in the Department of Biomedical Engineering at Texas A&M University are seeking to advance the field of 3D bioprinting functional tissues, by conducting research into the development of new biomaterials.  Dr. Akhilesh K. Gaharwar, an associate professor in the department, has created a highly 3D printable bioink, which can be used as a platform […]

Texas A&M Researchers Make “NICE” Bioinks to Create Functional Bone Tissues

Managing bone defects and injuries using traditional treatments can be slow and expensive. When bones break, bone cells can usually repair them, unless the break is too large. In that case, clinicians have historically turned to bone grafts using non-essential segments of bone taken from other parts of the same patient; bone from the hips, pelvis, chin, or ribs can do the job. Unfortunately, this requires additional surgeries, which translates into more pain for patients, and basically, there is a limit to how much non-essential bone surgeons can take from a patient.

As in many fields, bioprinting is disrupting the way healthcare specialists think about solving problems. So with an estimated 500,000 annual bone grafting procedures in the United States and more than 2 million around the world, an efficient bone substitute could change millions of lives. In a search to fabricate patient-specific, implantable 3D constructs for regenerative medicine, scientists in the Department of Biomedical Engineering at Texas A&M University have developed a new bioink formulation for 3D bone bioprinting called NICE, which is short for Nanoengineered Ionic–Covalent Entanglement, and have gone on to demonstrate that this bioink can precisely reconstruct large bone structures based upon CT scans that were obtained from actual patients.

Led by Akhilesh K. Gaharwar, an associate professor in the Department of Biomedical Engineering, the research group developed a highly printable bioink as a platform to generate anatomical-scale functional tissues. Their study was recently published in the American Chemical Society’s Applied Materials and Interfaces scientific journal, whereby they state that the NICE bioinks allow precise control over printability, mechanical properties, and degradation characteristics, enabling custom 3D fabrication of mechanically resilient, cellularized structures.

Bioprinting requires cell-laden biomaterials that can flow through a nozzle like a liquid, but solidify almost as soon as they’re deposited. This is why bioinks need to act as both cell carriers and structural components, requiring them to be highly printable while providing a robust and cell‐friendly microenvironment. However, the research group realized that many current bioinks lack sufficient biocompatibility, printability, structural stability, and tissue‐specific functions needed to translate this technology to preclinical and clinal applications.

To address this issue, Gaharwar and his team are leading efforts in developing the more advanced NICE bioinks, essentially a combination of two reinforcement approaches, ionic-covalent entanglement, and nanoreinforcement. In fact, the researchers claim that to design the NICE reinforced bioinks for osteogenic tissue bioprinting, the bioink must be highly printable, mechanically strong, induce osteogenic differentiation, and be biodegradable. However, the difficulty of combining these requirements into a single bioink has been a major obstacle in bioprinting since its inception. So by combining these two distinct reinforcement methods, NICE becomes a robust and superior bioink while providing a highly hydrated and cell-friendly microenvironment for bone bioprinting.

NICE printed structures are highly flexible and resilient, as seen in these 3D printed tube structures that can be completely collapsed and quickly regain their shape (Credit: Texas A&M Engineering)

According to Texas A&M Today, Gaharwar said that developing replacement bone tissues could create exciting new treatments for patients suffering from arthritis, bone fractures, dental infections, and craniofacial defects.

“The next milestone in 3D bioprinting is the maturation of bioprinted constructs toward the generation of functional tissues,” Gaharwar said. “Our study demonstrates that NICE bioink developed in our lab can be used to engineer 3D-functional bone tissues.”

NICE bioinks have three major components: covalently crosslinkable gelatin methacryloyl (GelMA), ionically crosslinkable kappa-carrageenan (kCA), and electrostatically charged nanosilicates (Laponite XLG, obtained from BYK Additives & Instruments).
To bioprint scaffolds that can demonstrate potential clinical uses, the team used their osteoinductive NICE bioinks. Then human mesenchymal stem cells (hMSCs) were encapsulated in the NICE bioink and bioprinted into 3D scaffolds. Once bioprinting was complete, this cell-laden 3D printed NICE structures were crosslinked to form stronger scaffolds. This technique has allowed the lab to produce full-scale, cell-friendly reconstructions of human body parts, including ears, blood vessels, cartilage and even bone segments.

hMSCs are encapsulated in the NICE bioink and cell-laden scaffolds are printed (Credit: Texas A&M Engineering)

Soon after bioprinting, the enclosed cells start depositing new proteins rich in a cartilage-like extracellular matrix that subsequently calcifies to form a mineralized bone over a three-month period. Texas A&M stated that almost five percent of these printed scaffolds consisted of calcium, which is similar to cancellous bone, the network of spongy tissue typically found in vertebral bones.

The scaffolds are initially transparent but turn translucent due to remodeling and deposition of nascent proteins after 60 days (Credit: Texas A&M Engineering)

To understand how these bioprinted structures induce stem cell differentiation, the team worked with Irtisha Singh from the Texas A&M Health Science Center, who served as a co-investigator, to use a next-generation genomics technique called whole transcriptome sequencing (RNA-seq) technology, which takes a snapshot of all genetic communication inside the cell at a given moment.
As for the bioprinting, the researchers modified a commercial ANET A8 3D printer kit to utilize screw extrusion. They replaced the thermoplastic extruder assembly with a 3D printed screw extruder assembly, which holds a stepper motor, guide rail, and a modified clay extruder.

To illustrate the practical utility of NICE bioinks for bone tissue reconstruction, the team demonstrated how to create full-scale bioprinted implants customized for craniofacial defects on real patient CT scans. Relying entirely on open-source software, they used the free 3D modeling software Meshmixer to process the models and create bone defects, and the 3D printing applications PrusaSlicer and Repetier Host to bioprint the scaffolds. After bioprinting, the scaffold was crosslinked and implanted in a thermoplastic model of the lower jaw to demonstrate the closeness of fit. Strength of fit was also demonstrated by injecting and crosslinking NICE bioink between two sections of a full-thickness fracture to prove that NICE is able to quickly adhere surfaces together and resist shearing and delamination forces.

Funded by the National Institutes of Health (NIH)’s Director’s New Innovator Award, a National Science Foundation (NSF)’s Award and an X-Grant from Texas A&M University, the researchers suggest they have discovered a new way to design and produce 3D bioprinted bone tissue to benefit bone regeneration.

Moreover, Gaharwar claims to have demonstrated that the highly printable NICE bioinks can precisely reconstruct large bone structures from CT scans obtained from actual patients. The aim of the research is to enable patient-specific bioprinting of bone scaffolds to precisely match their injuries. The researchers stated their desire to have this technique act as a customizable and easy to work with an alternative to autografts that will provide surgeons with greater options for bone surgery. And with the ultimate goal of getting NICE bioink technology from bench to bedside, Gaharwar’s team plans to establish the in vivo functionality of the 3D bioprinted bone tissue.

3D-bioprinted NICE scaffolds can be used for bone regeneration (Credit: Texas A&M Engineering)

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Texas A&M: A Method for 3D Printing Porosity Free Martensitic Steels

While seeking a corrosion-resistant alloy for gun barrels in 1912, British researcher Harry Brearley, who is commonly regarded as the inventor of stainless steel, discovered a martensitic stainless steel alloy. Although several variants of steel exist today, this type particularly stands out from its steel cousins as stronger and more cost-effective to produce. The renowned metallurgist probably never thought that his breakthrough discovery would go beyond developing affordable cutlery to the masses, well into applications in the aerospace, medical, automotive, and defense industries. Now over 100 years later, it can also be used as a metal 3D printing material for complex designs.

However, for these and other applications, the metals have to be built into complex structures with minimal loss of strength and durability, which is why researchers from Texas A&M University, in collaboration with scientists in the Air Force Research Laboratory, have developed guidelines that allow 3D printing of martensitic steels into very sturdy, defect-free objects of nearly any shape.

Reported in the scientific journal Acta Materialia, the findings of their study suggest that the process optimization framework introduced is expected to allow the successful printing of new materials in an accelerated fashion and introduces the process parameters for building porosity-free parts.

Although the procedure developed was initially for martensitic steels, the researchers said they have made their guidelines general enough so that the same 3D printing pipeline can be used to build intricate objects from other metals and alloys as well.

“Strong and tough steels have tremendous applications but the strongest ones are usually expensive — the one exception being martensitic steels that are relatively inexpensive, costing less than a dollar per pound,” said Ibrahim Karaman, Chevron Professor and head of the Department of Materials Science and Engineering at Texas A&M. “We have developed a framework so that 3D printing of these hard steels is possible into any desired geometry and the final object will be virtually defect-free.”

A flowchart summarizing the framework, introduced in this study (Credit: An ultra-high strength martensitic steel fabricated using selective laser melting additive manufacturing: Densification, microstructure, and mechanical properties)

The high-strength, lightweight, and cost-effective martensite steels are formed when steels are heated to extremely high temperatures and then rapidly cooled. The sudden cooling unnaturally confines carbon atoms within iron crystals, giving martensitic steel its signature strength.

Texas A&M claimed that to have diverse applications, martensitic steels, particularly a recently discovered type of low-alloy, ultra-high-strength martensitic steel known as AF9628, need to be assembled into objects of different shapes and sizes depending on the particular application they will be used for, and that’s when additive manufacturing (AM) offers a practical solution.

Stainless steels can be used to 3D print complex designs that are normally impossible to fulfill. 3D printing methods initially used by the team to build complex items were direct metal laser sintering (DMLS) aka selective laser melting (SLM) and also known as Powder Bed Fusion. However, Texas A&M researchers detected that 3D printing martensitic steels using lasers can introduce unintended defects in the form of pores within the material. Moreover, they detected that there is currently no known work describing process-structure-property relationships for AF9628 in the context of AM, something they considered should be systematically studied, focusing on the effects of AM process parameters on the microstructural evolution and resulting mechanical properties of this new martensitic steel.

“Porosities are tiny holes that can sharply reduce the strength of the final 3D printed object, even if the raw material used for 3D printing is very strong,” Karaman said. “To find practical applications for the new martensitic steel, we needed to go back to the drawing board and investigate which laser settings could prevent these defects.”

In an effort to produce high strength parts with a high degree of control over geometry, the researchers presented the effects of the SLM parameters on the microstructure and mechanical properties of the new steel AF9628.

For their experiments, Karaman and his team first chose an existing mathematical model, called Eagar-Tsai, inspired from welding to predict the melt pool geometry, that is, how a single layer of martensitic steel powder would melt for different settings for laser speed and power. By comparing the type and number of defects they observed in a single track of melted powder with the model’s predictions, they were able to change their existing framework slightly so that subsequent predictions improved.

They claim that after a few of these iterations, their framework could correctly forecast, without needing additional experiments, if a new, untested set of laser settings would lead to defects in the martensitic steel.

Raiyan Seede, a graduate student in the College of Engineering at Texas A&M and the primary author of the study, explained that “testing the entire range of laser setting possibilities to evaluate which ones may lead to defects is extremely time-consuming, and at times, even impractical. By combining experiments and modeling, we were able to develop a simple, quick, step-by-step procedure that can be used to determine which setting would work best for 3D printing of martensitic steels.”

Seede also noted that although their guidelines were developed to ensure that martensitic steels can be printed devoid of deformities, their framework can be used to print with any other metal. He said this expanded application is because their framework can be adapted to match the observations from single-track experiments for any given metal.

“Although we started with a focus on 3D printing of martensitic steels, we have since created a more universal printing pipeline,” Karaman indicated. “Also, our guidelines simplify the art of 3D printing metals so that the final product is without porosities, which is an important development for all type of metal additive manufacturing industries that make parts as simple as screws to more complex ones like landing gears, gearboxes or turbines.”

Backscattered electron images of the etched cross-sections of AF9628 ultra-high strength martensitic steel as-printed cubes. The yellow dotted lines indicate melt pool boundaries (Credit: An ultra-high strength martensitic steel fabricated using selective laser melting additive manufacturing: Densification, microstructure, and mechanical properties)

This research, funded by the Army Research Office and the Air Force Research Laboratory, reports a successful methodology to determine optimal processing parameters, like laser power, laser scan speed, and hatch spacing, in selective laser melting AM in order to fabricate porosity-free parts.

The team of researchers effectively used it to fabricate fully dense samples over a wide range of process parameters, allowing the construction of an SLM processing map for the new martensitic steel alloy AF9628. Given the potential of this new high-performance steel, useful for machine tool components, structural components for aircraft gear, automotive parts, and even for ballistic armor plates, creating a new framework offers the potential to 3D print this new material much quicker, providing a powerful tool to many industries.

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US Army Research Lab Scientists Creating Atomic-Level 3D Reconstructions of Specimens

The US Army Research Laboratory (ARL) is responsible for plenty of innovative 3D printing research over the years, such as 3D printing drones and working with recycled 3D printing material. Now, material scientists from the ARL have their sights set on something much smaller that could have a very large impact – analyzing atomic-level metal and ceramic specimens.

Dr. Chad Hornbuckle, a materials scientist with the ARL’s Weapons and Materials Research Directorate, specializes in microstructural characterization using electron microscopes and atom probe tomography (APT), and is working on the atomic-level research. He said that the unique atom probe being used in this research not only sets the standard for accuracy in chemistry, but is also necessary to understanding the interior structure of materials themselves.

“The atom probe gives us a 3-D reconstruction at the atomic level. When you see the reconstruction that’s made up of millions of dots, the dots are actually individual atoms,” Dr. Hornbuckle explained.

“It’s basically the only machine in the world that can do this at the atomic level. There are machines, like transmission electron microscopes, or TEMs, that do chemical analysis, but it is not as accurate as this.”

Dr. Chad Hornbuckle, a materials scientist with the ARL, specializes in atom probe tomography, which analyzes ceramics or metal 1,000 times smaller than a human hair.

Because experiments require consistency, it’s extremely important to maintain a high level of accuracy during research like this.

Dr. Hornbuckle said, “You might have one effect one time, but if the chemistry changes, you get a completely different effect the next time. If you can’t control the chemistry, you can’t control the properties.”

If you thought working at the nanoscale level was small, consider this: the atomic-level specimens being analyzed in this research are roughly 1,000 times smaller than the end of a strand of human hair. Researchers have to create very sharp tips to get the samples ready for analysis, which are used to mill, or sandblast, the materials away using gallium and either a focused ion beam microscope or a dual beam scanning electron microscope. Then they are inserted into the atom probe.

The interior of the probe is a super cold vacuum. Atom samples are ionized with a laser, or a voltage pulse, within the probe’s tip, which causes them to field evaporate from the surface. Then, the evaporated ions are analyzed and identified, which results in a 3D model with a near-atomic spatial resolution.

Atom probe

Dr. Hornbuckle himself developed the probe during his time as a graduate student at the University of Alabama. Army scientists and other researchers now ask him for his help in characterizing samples, and use APT technology to determine which atoms are located where in a material.

Dr. Denise Yin, a postdoctoral fellow at ARL, said, “I can give you one specific example of how it’s helped our research. We were electrodepositing copper in a magnetic field and we found a chemical phase using the atom probe that didn’t otherwise show up in conventional electrodeposition.

“Electrodeposition is a process that creates a thin metal coating.

We were having problems identifying this phase using other methods, but the atom probe told us exactly what it was and how it was distributed.”

Dr. Yin said that the atom probe has “impressive” capabilities:

“You can see the atoms show up in real time. Again, it’s at the nanometer scale, so it’s much finer than all the other characterization techniques. The atom probe told us quite easily that the unknown phase was two different types of a copper hydride phase, and that’s not something that we could have detected using those other methods.”

[Image: ARL]

Only a limited number of these atom probes exist, and the one used by the ARL is one of just several in the US. So you can imagine that many universities hope to use it to analyze their own samples. As part of its Open Campus business model, the lab looks for formal agreements.

ARL Director Dr. Philip Perconti explained, “Open Campus means sharing world-class ARL facilities and research opportunities for our partners. A thriving Open Campus program increases opportunities for technology advancement and the transfer of research knowledge.”

Dr. Hornbuckle said that a partnership with Lehigh University yielded some “important results.”

Army scientists explore materials at the nano-level with the goal of finding stronger or more heat-resistant properties to support the Army of the future.

“One university that we collaborate with is Lehigh University. At first, this collaboration was more of a mutual exchange of expertise, where I analyzed some of their samples in the atom probe and they used their aberration-corrected transmission electron microscope to analyze some of our copper-tantalum sample,” said Dr. Hornbuckle. “We now have a cooperative agreement with them to continue this collaboration.

“I actually ran a nickel-tungsten alloy that was electrodeposited for them and identified and quantified the presence of low atomic number elements such as oxygen and sodium. This resulted in one research journal article with several more in preparation.”

The ARL is also collaborating with Texas A&M University on atomic-level analysis.

“This collaboration initiated due to the Open Campus initiative. I have analyzed a few nickel-titanium alloys that had been 3-D printed. They noticed some nanoscale precipitates within the 3-D printed materials, but were unable to identify them with their TEM,” Dr. Hornbuckle said. “I am trying to determine the chemistry of the phase using the atom probe, which should help to identify it.”

The University of Alabama is another of the ARL’s partners, and this collaboration led to several published research journal articles.

“They have a different version of the atom probe. They have run some our alloys in their version and ours to compare the differences noted in the same material. This material is actually being scaled up through a number of processes that are relevant to the Arm,” Dr. Hornbuckle explained.

In addition to creating important and meaningful connections, these various partnerships also provide the Army with access to equipment not found at the ARL. Then, the knowledge that Army researchers learn through this joint research can be applied to current problems the Army is facing, as well as to developing future relevant materials.

Dr. Hornbuckle said, “When you see things no other human has ever seen before, it’s very cool to think that I’m helping to push the envelope of new modern materials science, which then obviously is used for the Army. Every time we run a new material we think about how we can help the Soldier with this new discovery.”

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[Images: US Army photos by David McNally]