August 19, 2013

At the Printer, Living Tissue


Published: August 18, 2013

SAN DIEGO — Someday, perhaps, printers will revolutionize the world of medicine, churning out hearts, livers and other organs to ease transplantation shortages. For now, though, Darryl D’Lima would settle for a little bit of knee cartilage.

Dr. D’Lima, who heads an orthopedic research lab at the Scripps Clinic here, has already made bioartificial cartilage in cow tissue, modifying an old inkjet printer to put down layer after layer of a gel containing living cells. He has also printed cartilage in tissue removed from patients who have undergone knee replacement surgery.

There is much work to do to perfect the process, get regulatory approvals and conduct clinical trials, but his eventual goal sounds like something from science fiction: to have a printer in the operating room that could custom-print new cartilage directly in the body to repair or replace tissue that is missing because of injury or arthritis.

Just as 3-D printers have gained in popularity among hobbyists and companies who use them to create everyday objects, prototypes and spare parts (and even a crude gun), there has been a rise in interest in using similar technology in medicine. Instead of the plastics or powders used in conventional 3-D printers to build an object layer by layer, so-called bioprinters print cells, usually in a liquid or gel. The goal isn’t to create a widget or a toy, but to assemble living tissue.

At labs around the world, researchers have been experimenting with bioprinting, first just to see whether it was possible to push cells through a printhead without killing them (in most cases it is), and then trying to make cartilage, bone, skin, blood vessels, small bits of liver and other tissues. There are other ways to try to “engineer” tissue — one involves creating a scaffold out of plastics or other materials and adding cells to it. In theory, at least, a bioprinter has advantages in that it can control the placement of cells and other components to mimic natural structures.

But just as the claims made for 3-D printing technology sometimes exceed the reality, the field of bioprinting has seen its share of hype. News releases, TED talks and news reports often imply that the age of print-on-demand organs is just around the corner. (Accompanying illustrations can be fanciful as well — one shows a complete heart, seemingly filled with blood, as the end product in a printer).

The reality is that, although bioprinting researchers have made great strides, there are many formidable obstacles to overcome.

“Nobody who has any credibility claims they can print organs, or believes in their heart of hearts that that will happen in the next 20 years,” said Brian Derby, a researcher at the University of Manchester in Britain who reviewed the field last year in an article in the journal Science.

For now, researchers have set their sights lower. Organovo, for instance, a San Diego company that has developed a bioprinter, is making strips of liver tissue, about 20 cells thick, that it says could be used to test drugs under development.

A lab at the Hannover Medical School in Germany is one of several experimenting with 3-D printing of skin cells; another German lab has printed sheets of heart cells that might some day be used as patches to help repair damage from heart attacks. A researcher at the University of Texas at El Paso, Thomas Boland, has developed a method to print fat tissue that may someday be used to create small implants for women who have had breast lumpectomies. Dr. Boland has also done much of the basic research on bioprinting technologies. “I think it is the future for regenerative medicine,” he said.

Dr. D’Lima acknowledges that his dream of a cartilage printer — perhaps a printhead attached to a robotic arm for precise positioning — is years away. But he thinks the project has more chance of becoming reality than some others.

“Printing a whole heart or a whole bladder is glamorous and exciting,” he said. “But cartilage might be the low-hanging fruit to get 3-D printing into the clinic.”

One reason, he said, is that cartilage is in some ways simpler than other tissues. Cells called chondrocytes sit in a matrix of fibrous collagens and other compounds secreted by the cells. As cells go, chondrocytes are relatively low maintenance — they do not need much nourishment, which simplifies the printing process

Keeping printed tissue nourished, and thus alive, is one of the most difficult challenges facing researchers. Most cells need to be within a short distance — usually a couple of cell widths — of a source of nutrients. Nature accomplishes this through a network of microscopic blood vessels, or capillaries.

But trying to emulate capillaries in bioprinted tissue is difficult. With his fat tissue, Dr. Boland’s approach is to build channels into the degradable gel containing the fat cells, and line the channels with the kind of cells found in blood vessels. When the printed fat is implanted, the tubes “start to behave as micro blood vessels,” he said.

The body naturally produces chemical signals that would cause it to start growing small blood vessels into the implant, Dr. Boland said, but the process is slow. With his approach, he said, “we expect this will be sped up, and hopefully keep the cells alive.”

With cartilage, Dr. D’Lima does not need to worry about blood vessels — the chondrocytes get the little nourishment they need through diffusion of nutrients from the joint lining and bone, which is aided by compression of the cartilage as the joints move. Nor does he need to be concerned with nerves, as cartilage lacks them.

But there is still plenty to worry about. Although it is less than a quarter of an inch thick, cartilage of the type found in the knee or hip has a complex structure, with several layers in which collagen and other fibrous materials are oriented differently.

“The printing demands change with every layer,” Dr. D’Lima said. “Most 3-D printers just change the shape. We are changing the shape, the composition, the type of cells, even the orientation of the cells.”

Dr. D’Lima has been involved in orthopedic research for years; one of his earlier projects, a sensor-laden knee-replacement prosthesis called the electronic knee, has provided invaluable data about the forces that act on the joint. So he was aware of other efforts to make and repair cartilage. “But we didn’t want to grow tissue in the lab and then figure how to transplant it into the body,” he said. “We wanted to print it directly in the body itself.”

He and his colleagues began thinking about using a thermal inkjet printer, in which tiny channels containing the ink are heated, producing a vapor bubble that forces out a drop. The technology is very reliable and is used in most consumer printers, but the researchers were wary because of the heat produced. “We thought it would kill the cells,” Dr. D’Lima said.

But Dr. Boland, then at Clemson University, and others had already done the basic research that showed that the heat pulse was so rapid that most cells survived the process.

Dr. D’Lima’s group soon discovered another problem: the newest thermal inkjets were too sophisticated for their work. “They print at such high resolution that the print nozzles are too fine for cells to squeeze through,” he said.

They found a 1990s-era Hewlett-Packard printer, a Deskjet 500, with bigger nozzles. But that printer was so old that it was difficult finding ink cartridges; the researchers finally located a supplier in China who had some.

Their idea was to replace the ink in the cartridges with their cartilage-making mixture, which consisted of a liquid called PEG-DMA and the chondrocytes. But even that created a problem — the cells would settle out of the liquid and clog the printhead. So the researchers had to devise a way to keep the mixture stirred up.

The mixture also has to be liquid to be printed, but once printed it must become a gel — otherwise the end product would just be a watery mess. PEG-DMA becomes a gel under ultraviolet light, so the solution was to keep the print area constantly exposed to UV light to harden each drop as it was printed. “So now you’re printing tissue,” Dr. D’Lima said.

But Dr. D’Lima and his group are investigating other materials for their gel. While PEG-DMA is biocompatible (and approved for use by the Food and Drug Administration), it would remain in the body and might eventually cause inflammation. So they are looking for substances that could degrade over time, to be replaced by the matrix produced by the chondrocytes. The printed material could be formulated to degrade at the same rate as the natural matrix is produced.

There are plenty of other challenges as well, Dr. D’Lima said, including a basic one — how to get the right kinds of cells, and enough of them, for the printer. It would not make much sense to use a patient’s own limited number of cartilage cells from elsewhere in the body. So his lab is investigating the use of stem cells, precursor cells that can become chondrocytes. “The advantage of stem cells is that it would mean a virtually unlimited supply,” Dr. D’Lima said.

Dr. D’Lima’s team is investigating other technologies that might be used in combination with bioprinting, including electrospinning, a method of creating the fibers in the matrix, and nanomagnetism, a way to orient the fibers. His lab takes a multidisciplinary approach — he even attends Siggraph, the large annual computer graphics convention, to get ideas. “They’re like 10 years ahead of medical technology,” he said.

Meanwhile, the lab has upgraded its printing technology. The Deskjet is still around, but it has not been used in more than a year. It has been supplanted by a much more sophisticated device from Hewlett-Packard — essentially a programmable printhead that allows the researchers to adjust drop size and other characteristics to optimize the printing process.

Dr. D’Lima said the biggest remaining hurdles were probably regulatory ones — including proving to the F.D.A. that printed cartilage can be safe — and that most of the scientific challenges had been met. “I think in terms of getting it to work, we are cautiously optimistic,” he said.