Fresh prints: the road to 3D printed organs

Darcy Wagner is on a quest to improve research into the human lung – and to develop synthetic therapies for both acute and chronic lung disease.

“These are the third and fourth leading causes of death in the world – and have no cure,” says Wagner, associate professor in the department of experimental medical science at Lund University in Sweden.

Patients with chronic lung disease have one option: a lung transplant. However, survival times are short compared to, say, a heart transplant, and there is an acute shortage of lungs for transplantation. These factors inform Wagner’s work in developing ways to produce synthetic lung tissue using 3D printing.

Now, it is being intensively researched – by Wagner and many others – as a way of manipulating biological material. Shortages of donor organs, and the ever-present chance of tissue rejection, are some of the factors behind this intense focus, though researchers accept that they are only at the start of a very long journey.

“3D bioprinting is a good platform to build on,” says Wagner. “I have high hopes it can make a difference and I can see it being used in patients.”

Wagner and her research team have developed a bio-ink that specifically mimics the structure of lung tissue. They have used the ink to 3D-print small airways such as those found in a human lung. The ink formulation helps to ensure that the tissue will not be rejected when it is implanted. The work was published recently in Advanced Materials.

“We’re not ready to print organs yet, but we’ve printed these small tubes,” she says. The new ink relies partly on alginate, a derivative of seaweed that is well established in tissue culture work. However, a second key ingredient is the extracellular matrix (ECM). This is the scaffold structure left behind when lung tissue has been ‘decellularised’. It is pulverised and mixed with the alginate to form the ink, and is critical in preventing an immune response.

Alginate gelates instantly with the addition of calcium ions, and also undergoes shear thinning, so becomes less viscous when compressed. The combination of ECM and alginate has higher shear thinning than alginate alone. “This means it takes less force to push it out of the nozzle – so there are lower shear forces to harm the cells,” says Wagner. “When we bio-print, the cells are contained within the ink.” The ink returns to higher viscosity once it emerges from the nozzle.

Wagner’s team makes its parts using a technique called FRESH, developed at Carnegie Mellon University in the US. This allows two types of cell to be printed at the same time – such as airway cells on the inside, and muscle cells on the outside. “A combination of FRESH and our ink allows us to do this,” she says.

The ink has been tested on mice by implanting 3D-printed discs of the material under the skin and then observing the effect. “If you use an implant made from alginate alone, you get an immune response,” she explains. “But our bio-ink had no immune response, and actually promoted healing.”

In addition, the team saw that new blood vessels grew in and around the implant. Both effects would be critical in ensuring that implanted parts are not rejected, and could help to prolong the lifespan of lung transplant patients. “Encouraging blood vessels to grow naturally is much better than having to print them,” says Wagner.

Any kind of production process – no matter how small the part produced – aims for fast throughput. Researchers at the University at Buffalo in the USA have developed a technique that makes relatively large biostructures while claiming to be much faster than other 3D-printing methods.

Using a modified version of stereolithography, the researchers have printed demonstration parts in less than 20 minutes. At the same time, the parts incorporate a crucial microstructure: embedded vessels, which could mimic the function of blood vessels. “It does not rely on a ‘dot by dot’ approach of a nozzle,” says Chi Zhou, associate professor of industrial and systems engineering at the university. He is co-lead author of a recent paper in Advanced Healthcare Materials.

Instead, the method – which the researchers call FLOAT (for fast hydrogel stereolithography printing) – creates “centimetre-sized” 3D structures within a bath of carefully selected liquids. The bath holds a circulating mixture of water, pre-polymer, photo-initiator, photo-absorbing dye and living cells. The pre-polymer is ‘cured’ using light, which turns it from liquid into a solid ‘hydrogel’ 3D part. Liquid circulation is critical to replenishing the supply of pre-polymer, ensuring that polymerisation is smooth and continuous.

Getting the mix of materials in the bath is crucial, says Zhou. It must have a low enough viscosity to flow freely. At the same time, the level of photo-absorbing dye must be carefully controlled: it is needed in order to ‘cure’ the part – and improve accuracy – but too much could harm the living cells. “If you don’t have the dye, the light will pass straight through,” he says.

A key differentiator for the technique, Zhou explains, is that living cells can be incorporated within the 3D structure, rather than only on the surface. “Usually, 3D scaffold parts are printed, and cells incorporated afterwards,” he says. “In real human tissue, cells are not just on the surface; they are encapsulated in the scaffold. We’ve simulated this exactly by mixing living cells in with the pre-polymer.”

The team has also taken steps to ensure that the cells are not destroyed by the 3D-printing process. Firstly, UV light – which is typically used for photo-polymerisation – has been replaced with ‘near blue’ light. This sits between UV and blue on the electromagnetic spectrum and is less harmful to cells. At the same time, the speed of the process means that cells are subject to 3D-printing conditions for a relatively short period, exposing them to less stress.

The researchers have so far used fibroplasts, which are commonly used in research, but have also used cardiac cells.

Another challenge for 3D-printed tissue is that nutrients must be provided to the cells. In the body, this is done via tiny blood vessels. In a printed structure, this is more difficult to achieve.

Ruogang Zhao, associate professor of biomedical engineering at Buffalo, and co-lead author of the paper, adds: “Blood vessels are the biggest challenge in tissue engineering. You can produce these using 3D printing, but it’s very difficult.”

He says that the new technique can produce the 3D ‘organ’ structure and the hollow channels at the same time. “We can design the geometry of the hollow channels into the material.”

The researchers printed and tested two types of part, which incorporated liver cells. One part was solid, with no vessels; the other included hollow channels and was infused with nutrients. The nutrients helped to maintain cell growth. “In contrast, the solid structure saw massive cell death,” says Zhao.

The researchers have already begun to commercialise their technique by applying for a patent and forming a start-up company. They say that potential ‘customers’ will be research departments in universities and the pharmaceutical industry.

“The final end-point is the clinical use of printed tissues for injury repair,” says Zhao. “The main motivation is the shortage of organs and donor tissue.” However, full commercialisation, where these parts are used in surgical procedures, is still around 20 years away, in Zhao’s estimation.

Researchers in Australia have taken a new approach to printing 3D ‘scaffold’ structures that could be inserted into the body to promote tissue regrowth.

Rather than printing the structures directly, researchers from RMIT University first print a miniature ‘mould’ and inject it with biocompatible material. The mould is then dissolved in water, leaving the scaffold behind. This, says researcher Cathal O’Connell, allows very intricate structures to be produced in biocompatible material, which cannot always be made using 3D printing.

“With 3D printing, the resolution is typically limited by the diameter of the nozzle,” he says. “In our case we define our pattern in the spaces between the extruded lines.”

O’Connell is the corresponding author of a recent paper in Advanced Materials Technologies which describes the technique. “It’s a bit like trying to draw a picture using a thick marker pen,” he explains. “The lines that you draw are as thick as the pen, but the space between them can be much thinner.”

The team has created a number of 3D structures, including one in the shape of an ear, with diameters of 0.12mm. This is less than one-third the size of the printing nozzle and about the width of two human hairs. The moulds are made from polyvinyl alcohol (PVA) filament, a water-soluble material that is available off the shelf.

“We pressure cast or inject a biocompatible material into the mould, then dissolve the mould with a benign wash,” O’Connell explains. He says it is important that mould removal is as gentle as possible because some of the biomaterials are quite delicate. “We also don’t want to use any nasty solvents that might cause toxicity issues down the track,” he says.

The paper focuses on the use of a biodegradable polymer called polycaprolactone. It is used clinically to make degradable sutures and commonly employed in tissue engineering and regenerative medicine as a scaffold. The team has also tested other materials, including a hydrogel called agarose, which is used as an environment for 3D cell culture in tissue engineering, and a mixture of polycaprolactone and ceramic hydroxyapatite, which is of interest in orthopaedics.

The printing technique itself, dubbed NEST3D (Negative Embodied Sacrificial Template 3D), is a modification of fused deposition modelling (FDM). O’Connell says FDM is the most common 3D-printing technique used in rapid prototyping. It works by extruding heated thermoplastic through a nozzle and stacking patterns layer-by-layer to make a 3D object.

However, the need to stack the layers can limit design freedom at the microstructural level, says O’Connell. At the same time, it is designed for thermoplastics, which can limit the choice of materials.

“By combining FDM with injection moulding, we can overcome these issues and create intricate scaffolds for use in bio-fabrication,” he says. The technique is now at proof-of-concept stage and has mainly been used to print test structures.

“Our main aim is to make highly porous, biodegradable implants,” O’Connell continues. “These could help to regenerate bone following cancer surgery, or knee cartilage, to prevent the onset of osteoarthritis.” He says the technique could be used in plastic surgery, for parts such as ears and noses, or for personalised medical implants. “For this, you need to control the architecture at the microscale, which is very difficult using the regulatory approved implant materials available today”.

When 3D printing took its first tentative steps in engineering, it revolutionised the production of design prototypes. 3D bioprinting is still in its infancy, but as it develops it could one day help to balance the mismatch between the supply and demand for organs.