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Printing Muscle and Bone

Ink-jet printers allow tissue engineers to control cell development and could one day be used to construct complex cellular structures.
December 19, 2006

Researchers at Carnegie Mellon University and the University of Pittsburgh have successfully directed adult stem cells from mice to develop into bone and muscle cells with the aid of a custom-designed ink-jet printer. They say it’s a first step toward better understanding tissue regeneration, which may one day lead to therapies for repairing damaged tissues, as occurs in osteoarthritis.

Carnegie Mellon scientists have successfully differentiated stem cells into two different lines using “bio-inks.” Phil Campbell and his colleagues first loaded a modified ink-jet printer with a bio-ink solution of growth factor GMP-2, known for turning stem cells into bone cells. Then, after printing squares of various shades, or layers, onto a glass slide, researchers coated it with muscle-derived adult stem cells. What they found: stem cells that landed within the squares differentiated into bone cells, and those that appeared outside the squares turned into muscle cells.

For years, tissue engineers have used souped-up printers, and in some cases off-the-shelf models, to print “bio-inks.” These inks consist of anything from proteins to individual cells printed in microscopic patterns. By printing layer upon layer of cell patterns, scientists may one day be able to “print” whole tissues or organs for replacement therapies.

Now Phil Campbell and his team at Carnegie Mellon have added a new branch to the budding field of bioprinting. Certain growth factors spur stem cells to morph into specific kinds of cells, such as bone or muscle. Campbell and his colleagues have successfully printed growth-factor solutions on the same slide, or “paper,” forming a scaffold onto which stem cells can interact and differentiate into bone or muscle cells side by side.

The team loaded its ink-jet printer with a dilute solution containing the growth factor BMP-2, known for turning stem cells into bone cells. Meanwhile, the researchers prepared the paper they would print on: a microscope slide coated with a fibrin matrix–a material found in the body that naturally binds growth factors. The team then printed growth factors one drop at a time, in four separate square patterns of 750 microns. Each square consisted of varying shades, or concentrations, of growth factor, depending on the number of times the researchers printed on top of the same pattern.

Once the slide was dry, researchers placed it in a culture dish and evenly coated it with adult stem cells taken from the leg muscles of mice. Stem cells landing on areas with growth factors began to differentiate into bone cells–the greater the concentration of growth factor, the higher the yield of differentiated bone cells. Stem cells that landed on blank spots turned into muscle cells, which is the default developmental path for these cells.

Campbell’s technique diverges from previous research, in which different types of stem cells are grown individually in separate flasks or incubation vessels. He says that being able to differentiate multiple kinds of cells, such as bone and muscle, side by side mimics the way stem cells naturally differentiate within the body.

“We’re recreating microenvironments that better replicate those that nature normally makes,” says Campbell. “You can envision a scaffold structure where one end promotes bone, one end tendon, the other end muscle. That gives you more control over regenerating that tissue.”

Researchers like the University of Missouri’s Gabor Forgacs who have worked in bioprinting see these findings as an interesting though preliminary application. “What is important about this work is they show in vitro that by changing the pattern or the concentration of this [growth factor], cells respond differently and choose their lineages according to the concentration,” says Forgacs. “Controlling stem-cell specification is very important, and we’re still at the beginning of this endeavor.”

Ink-jet printing may ultimately open up a whole new way of studying stem cells. Lee Weiss, a research professor at Carnegie Mellon’s Robotics Institute who helped design the custom printer, says the technology is there; the question is how to better use it.

“I can print fairly complex things,” says Weiss. “But probably one of the biggest limiting factors is understanding the biology in order to know what to print.” Weiss hopes to eventually print specific patterns of growth factors that can then either be combined with stem cells in vitro or implanted directly into damaged regions to create new tissue.

To that end, researchers are now exploring even more complex patterns and printing with other growth factor-based “inks,” all with a view toward tissue therapy. Campbell adds that to take this to the next level, they are also working on 3-D patterning, which may be used to direct stem-cell transplantation. For now, Campbell is looking to experiment with other types of stem cells, including those found in human adults.

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