The scientists first analysed a digital 3-D image of a human ear, then used a 3-D printer to assemble a mould, into which they injected a mixture of collagen gel from rat tails and cartilage cells from cow ears. The collagen served as a scaffold upon which cartilage could grow, and over three months, the cartilage grew to replace the collagen.
"It takes half a day to design the mould, a day or so to print it, 30 minutes to inject the gel, and we can remove the ear 15 minutes later. We trim the ear and then let it culture for several days in nourishing cell culture media before it is implanted," says Professor Lawrence Bonassar, leader of the study.
The bioengineered ear replacement may be the solution reconstructive surgeons have long wished for to help children born with ear deformities, says co-lead author Dr Jason Spector. Microtia, a congenital deformity where the external ear is not developed, affects thousands of children each year. Many people also lose part or their entire ear in an accident or from cancer.
Three-dimensional printing technology is already generating revenue in two areas: dental fabrication of crowns, bridges, and implants; and prosthetics manufacturing.
Last month, an 83-year-old Belgian woman, whose chronic bone infection had destroyed her lower jaw, was able to eat and speak again with a 3-D printed prosthetic jaw made from 33 layers of titanium powder that were heated, fused together and then coated with bioceramic artificial bone.
The area of "bioprinting" - the 3-D printing of human organs for transplant - however, is still in its infancy. Bioprinters use a "bio-ink" made of living cell mixtures to build a 3-D structure of cells, layer by layer, to form tissue. This tissue is then developed into organs.
Liver tissue, heart tissue, and pancreatic tissue will continue to be areas for us to explore
Dr Jordan Miller, University of Pennsylvania
In 2011, a team led by Anthony Atala, at the Wake Forest Institute for Regenerative Medicine in the US, revealed the development of a technique to grow engineered urethras for several Mexican boys whose urethras were damaged in car accidents. The scientists took a sample of tissue from the boys and multiplied the cells in the lab before seeding them on a cylinder of biodegradable material. The resultant tube of tissue was transplanted into the boys' urinary systems.
"When they came in, they had a leg bag to drain their urine, and they had to carry it everywhere they went," says Atala. After the treatment, "these children are now normal".
Replacing human body parts that are primarily made of cartilage, such as joints, the trachea and the nose, is helped by the fact that cartilage does not require a blood supply to survive. Building organs that rely on blood is trickier - though University of Pennsylvania scientists have been making advances in this area.
In a study published last year in the journal Nature Materials, the scientists showed that 3-D printed templates of filament networks can be used to rapidly create vasculature and improve the function of engineered living tissues. Without a vascular system, which delivers nutrients while removing waste products, living cells on the inside of a 3-D body part cannot survive.
Building a vascular network is tricky because the layer-by-layer fabrication of 3-D printing creates structural seams between the layers, which could burst when fluid is pumped through them at high pressure - as in the body's blood vessels.
The researchers designed 3-D filament networks in the shape of a vascular system that sat inside a mould. The mould and the vascular template were removed once cells were added to form a solid gel tissue around the filaments.
To find the optimal material, the team tested different formulations using a simple material: sugar. Sugars are mechanically strong, so the printed 3-D network could be sufficiently rigid to support its own weight. In addition, the template could be easily dissolved and flow out of the gel construct, leaving a vascular architecture spread throughout the bioengineered tissue.
"The perfect cylinders we are moulding into engineered tissues are similar to those that make up human blood vasculature," says Dr Jordan Miller, the study's lead author.
To print the sugar network, the scientists modified a commercially available 3-D printer called RepRap to extrude molten sugar with high precision. Once the template was made, gels containing human cells were set around the sugar network to create tissue. The sugar was then dissolved, leaving a channel through which nutrient-rich media could be pumped to feed the cells inside.
The team created a piece of liver tissue using human liver cells. "Our 3-D culture technique is able to create tissues in physiologically relevant architectures and at normal cell densities - tens to hundreds of millions of cells per millilitre - which allows us to explore cellular behaviour in a way that more closely mimics how cells grow in the body," says Miller.
Armed with this new technology, Miller and his colleagues are studying future possibilities. "We are investigating using this technique for making large-scale engineered tissues containing tens of millions of cells," he says. "Liver tissue, heart tissue, and pancreatic tissue will continue to be areas for us to explore."
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