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Highly customizable 3D-printed material induces bone regeneration

By Joseph Bennington-Castro November 4, 2016

To repair bone defects, scientists in the last two decades have focused a lot of attention on creating synthetic bone grafting

Hyperelastic bone
Photograph of a “hyperelastic bone” of human spinal section (Lumbar 1-5), which was three-dimensionally printed at room temperature with a specialized ink with a 9:1 ratio of hydroxyapatite and polymer. Credit: Adam E. Jakus, PhD

products, particularly those made from bioceramics and bioceramic composite constructs. Yet, even the best among these advanced scaffolds are not ideal, requiring, for example, high temperature techniques in excess of 100°C, which precludes the incorporation of biological factors and molecules. But researchers at Northwestern University have now created highly customizable “hyperelastic bone” scaffolds, which are three-dimensionally (3D)-printed at room temperature and can be shaped and deformed to fit wherever they are needed in the body. Made from a composite of bioactive ceramic and biodegradable polymer, the biocompatible material induces bone regeneration and, as reported in Science Translational Medicine, was able to effectively fuse vertebrae in rats and repair the damaged skull of a macaque monkey.

“Hyperelastic bone is a unique type of material that has very exciting properties that haven’t been seen with this type of ceramic and polymer composite before,” says study principle investigator Ramille Shah, a Northwestern materials scientist. “Surgeons really haven’t seen these types of properties in scaffolds before and they are excited to [eventually] put them into patients.”

Today, autografts—a graft that uses bone from another part of a patient’s body—are commonly used to repair bone defects, such as shattered or missing bones. But this painful procedure can leave defects from where graft material is removed. Also autografts are not easily applicable for fixing bone defects in places like the face, which has distinctive curves and shapes. “Those limitations would be addressed if we had a synthetic material that could be 3D-printed to match the defect space and fit perfectly,” Shah says.

Such synthetic materials often include ceramics based on calcium phosphate (CaP), a bioactive substance that is the main constituent of bones and teeth. These products, which come in different formulations such as granules and malleable putties, have limited use in the clinic today with several notable deficiencies, including being stiff and brittle (making them difficult to implant) and potentially causing inflammation if the body rejects the material or infection if the material does not degrade as quickly as the body needs it to. Moreover, sometimes the scaffolds just do not work, with the stem cells that accumulate on the scaffolds being unable to synthesize new bone.

More recent technologies have focused on composites of hydroxyapatite (a type of CaP mineral) that can be 3D-printed with established ceramic additive manufacturing techniques. These advances have increased mechanical elasticity over pure CaP products but the materials can still undergo brittle fracture. They are also fabricated with hot-melt fused deposition modeling or laser sintering techniques, which are not only too slow for mass fabrication, but also prevents incorporating things such as growth factors for improved bone regeneration and yields products with low bioactivity unless costly surface modifications are used. “We wanted to take the heating element out of it and find a composite that is printable to address these issues,” Shah says.

As with some previous high-temperature 3D-printable composites for bone, Shah and her colleagues began with hydroxyapatite and a biocompatible polymer already used in medicine—either polycaprolactone (PCL) or poly(lactic-co-glycolic acid) (PLGA). But to print at room temperature, they needed to use the correct solvent system, so study first author and Northwestern postdoctoral fellow Adam Jakus delved into the scientific literature for different solvent systems used for 3D-printing. He found a promising tri-solvent system comprised of the evaporate dichloromethane, surfactant 2-butoxyethanol, and plasticizer dibutyl phthalate.

The team mixed the three ingredients together into an ink—with a 9:1 ratio of hydroxyapatite:polymer—and printed it at room temperature, and found that the material was highly elastic and malleable. A 3D-printed sheet of hydroxyapatite and PLGA could be rolled, folded into origami shapes, and cut, for example. “We were not expecting that it would have such elastic properties,” Shah explains. They were also able to print highly specific shapes, such as a human mandible, as well as more complex structures by fusing together multiple printed objects (using the ink as a “glue”).

After looking at the microstructure of their hyperelastic bone, the team found that one of the keys to their material’s elasticity is its 50% inherent porosity, which allows particles to move into the voids during deformation. Interestingly, increasing the amount of polymer decreases the porosity, making the material more difficult to deform and increasing its stiffness. Though highly elastic, the biomaterial can be printed into architectures that are also strong—a 3D-printed femoral bone could withstand a load of 150 pounds. The team also compared hyperelastic bone that uses PLGA with one that uses PCL, and found that the PLGA version was easier to print, more flexible (withstanding 60% tensile strain compared with the PCL version’s 35%), and degraded more quickly. But, Shah notes, that does not mean the PLGA version is best for all circumstances, as there may be cases where a stiffer construct or prolonged degradation times is needed.

When the hyperelastic bone is placed in a body, cells recognize the hydroxyapatite as a natural bone or perhaps an incomplete bone that needs to be remodeled. Stem cells receive signals from the hydroxyapatite to begin synthesizing a bone matrix or remodeling the scaffold. Next, blood vessels start to penetrate and grow in the porous scaffold. Over time, the polymer degrades and is replaced by real tissue and the hydroxyapatite is remodeled into natural bone. 

Shah, Jakus, and their colleagues tested hyperelastic bone in two animal models. In the first, they used their technology to fuse two spinal vertebrae in a rat. After 4 weeks, the scaffold was infiltrated by surrounding tissue and blood vessels; after 8 weeks, the new bone formation was comparable to a control that used a cadaver-derived demineralized bone matrix scaffold, which is commonly used in bone repair. This demineralized bone matrix, however, is relatively expensive and carries the risk of disease transmission and has variable efficacy (depending on the quality of bone sources).

In the second model, the team sought to repair the skull of a rhesus macaque that had a large region of weak cranial cortical bone. Not knowing the exact size and shape of the defect, they printed out a sheet of hyperelastic bone similar to the structure of the cranium, which they were able to cut to size and press into the defect space. “This really shows the ability to scale to a relevant size and manipulate the shape and structure as needed in surgery,” Shah says. After a month, the scaffold was infiltrated with new blood vessels, tissue, and the beginning of new natural bone.

“The in vivo results seem to be very promising just by using simple materials without growth factors and further functionalization,” says Piergiorgio Gentile, a biomedical engineer at Newcastle University in the United Kingdom. Gentile, who was not involved in the study, was very surprised by the material’s high tensile strength and liked its use of simple components.

Chris Arts, a translational biomaterials researcher at Maastricht University Medical Centre in the Netherlands, was impressed by the work, in particular the technology’s wide-ranging applications and ease of production. But he notes that the material needs to be validated in large animal models in load-bearing defects. “The degradation process and speed of degradation in the body also needs to be studied to make sure no inflammation will occur over time,” he says.

Shah and her team are currently working toward beginning clinical trials, which they hope to achieve within the next five years. They are also looking into using their technology—ink systems containing biomedical elastomers—for non-biomedical applications and have already expanded their portfolio to include metal oxide (ceramic) and metallic inks, which could be useful for everything from energy to electronics to space (using Martian soil to make something functional, for example). “This technology really widens the playing field for what can be created with extrusion-based printing and makes it more accessible to a lot of people,” Shah says.

Read the abstract in Science Translational Medicine.