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FROM GENESIS TO REGENERATION
Engineering Tissue to Rebuild the Body
BY KATHLEEN YOUNT

When a cell is born, it usually knows just what it’s meant to be. In that moment when a single cell divides to become two new cells, all the proper genes for all the proper proteins are turned on, all the unnecessary genes are turned off, and the daughter cells set about living their lives as blood cells, muscle cells, heart cells, and so on.

There are some cells, however—such as certain cells in our bone marrow—that haven’t necessarily decided what form they will take. These cells, called mesenchymal stem cells, are what scientists call pluripotent. Their genes can be prodded and persuaded to turn on or off in new patterns, to make the cells into any of many cell types. These stem cells in our bone marrow could fashion themselves into new bone marrow, but they could also be taught to become cartilage, skeleton, even heart muscle cells.

Bioengineer Timothy Wick, Ph.D., is in the business of coaxing various kinds of cells toward new destinies, to help our bodies better heal after injury or repair tissue that’s been damaged by disease. As the new chair of UAB’s Department of Biomedical Engineering, recruited last year from the Georgia Institute of Technology, Wick is partnering with basic researchers and clinicians in the Schools of Medicine and Dentistry to make UAB a central figure in the field of regenerative medicine.

Made from Scratch

“The idea of tissue engineering is to make living replacement parts that function biologically like the parts they are replacing,” Wick says. “To be successful, such tissue will have to live and grow in the body, and it’ll have to function like the normal tissue we are born with.” It’s simple to say, yet sounds like the most mind-boggling feat of futuristic medicine. But if scientists have already found a way to grow skin tissue—physicians use it to graft onto severely burned or wounded skin—then could an engineered pancreas really be that far behind?

Wick says research is moving forward on several tissue-engineering fronts. Scientific teams around the world are creating blood vessels, heart muscle, bones, cartilage, tendons—efforts are even under way to grow nerves and entire organs such as the pancreas, kidney, and liver. With each type of tissue, these scientists are somewhat like master chefs trying to re-create a complex entrée without access to the original recipe. They experiment with different ingredients—trying to determine what kind of cells should be combined with what sort of growth-stimulating nutrients, on what kind of cellular matrix or scaffolding, and how best to “cook” the resulting biologic soup in order to create bones that will be strong or blood vessels that will reliably carry blood through the body. Many questions must still be answered before human trials of engineered tissue can be conducted, but Wick says many of these endeavors are moving closer and closer to clinical testing. He thinks it’s possible that in five to 10 years some sort of engineered tissue will be well on its way to use in medical care.

The Finest Cookware

Wick may work in biomedicine but he’s still an engineer at heart, so he approaches the coming wave of regenerative medicine from an engineer’s perspective. “The demand for some of these therapies in America will be on the order of 100,000 per year,” he says. “Some could even be a million a year—cartilage replacements or coronary arteries for bypass surgeries, for example.”

Any medical miracle demanded in those quantities will need a well-engineered system of production. That’s where Wick comes in. “Beyond thinking about the basic science of how to make a bone versus an artery or any replacement part, we also have to think about the questions of scale-up,” he says. “When we find something that works, we’re no longer going to be talking about a benchtop effort—a grad student or a postdoc in a lab making one or two at a time. We’re going to need large physical manufacturing facilities, not unlike facilities that manufacture auto parts. How do you manage all of that, and how do you make it profitable so that companies will be willing to invest in it?”

Wick believes the answer is to invent a fundamental tissue production unit that can be built with relative ease and housed in a manufacturing plant. Such a machine, he says, must be able to provide not only the right combinations of nutrients, but also mechanical stimulation to make tissue grow. “The reason bone becomes bone and heart becomes heart is that the stem cells get different chemical signals that tell them, ‘make a hunk of heart tissue’ or ‘make a piece of bone,’” says Wick. “So we have to provide that for the cells—with a sort of broth of nutrients that includes these chemical signals as well as things like oxygen.

“But you also need mechanical forces to make tissue,” he adds. “Part of the function of each tissue is mechanical—bone provides load-bearing structure, cartilage provides lubrication, blood vessels hold pulsing blood. So there are forces that are constantly acting on each type of tissue—and, as it turns out, those same forces actually help grow that tissue. In other words, in order to make a working blood vessel, you have to provide the shear forces of flow to the cell culture; in fact, you probably need to provide pulsing flow, just like that of moving blood.”

To do this, Wick has designed a device called a bioreactor, which serves as both the pot to hold this tissue-brewing stew and the whisk that constantly and consistently stirs the nutrient broth as it grows into functioning tissue. There are several bioreactors in development and use around the world; Wick has brought a few to UAB that he designed at Georgia Tech, and he’s now building new bioreactors with modifications that he’s been developing over the last few years. His main focus is on cartilage and blood vessels, and he creates specialized bioreactors for each kind of tissue.

The prototype bioreactor that Wick designed for cartilage construction comprises two small cylinders; the outer cylinder is about six inches tall and six inches in diameter. Inside the outer container is a smaller cylinder. Ten to 20 discs of stem cells are put into the bioreactor and adhere to the inner cylinder. The outer cylinder rotates to provide the mechanical stimulation, and the region between the two is filled with the nutrient soup. Right now it takes about six weeks to culture the cell discs into viable cartilage cells; Wick’s team monitors them throughout the process.

“We built the bioreactor to be controllable,” he says. “We know everything that happens at every point in the bioreactor at every instant in time, so we can change conditions and get predictable results. We can say, ‘Well, this part of the tissue doesn’t look very good, so let’s go back and turn these dials. Let’s put more of this, or less of this, or apply this force longer, or provide more oxygen.’ And we can then systematically solve problems of poor performance.”

A Complicated Cookie

The bioreactor is designed to perfect techniques for mass tissue production and even storage of engineered tissue. But Wick says the bioreactor also helps move research in tissue regeneration forward on a basic-science level. “That’s because we can put different chemicals into it, experiment with different mechanical forces, and really identify what optimizes tissue growth,” he says. This will be particularly useful in answering a major question that perplexes tissue engineers: how to build tissue that, in nature, has different cell types at different places within its structure.

“Most tissue is polar,” Wick explains, “and that means it’s different on different sides. The inside of a blood vessel has special kinds of cells that prevent clotting—the endothelial cells. But the vessel walls have muscle cells that control the contraction of the vessel. These are very different kinds of cells. A heart-valve leaflet, though it’s very thin, has three distinct layers that have three distinct compositions—and it’s the integrative function of those three layers that gives the valve the ability to flap thousands of times a day. With cartilage, one surface is smooth, because it lubricates the intersection between two bones. The other side is connected to a bone. So that tissue gradually changes from cartilage-like tissue to very bone-like tissue.

“Everyone’s initial attempts at tissue engineering have been to try to make tissue that is fairly consistent in its makeup. But many of us are starting to think that we need to be able to make tissue that is not so homogeneous.”

So how, exactly, do these scientists plan to cook up tissue structures that have multiple cell types? “Well, we don’t know yet,” says Wick. But he believes the bioreactor can be designed even for this delicate process. “We’ve developed some bioreactors in which we can change the flow on one side versus another side, to provide different mechanical stimulation to the cells on each side.” For example, his newest bioreactor for cartilage replaces the inner cylinder with a tube-like structure, so that the tissue disc rests on the open end of the tube. That way one side of the tissue disc can be exposed to the nutrients and flow in one compartment, while the other can be exposed to different nutrients and flow forces within the tube.

“And if we have different flows on different sides, we can deliver different nutrients with each flow,” he says. “So in the case of cartilage, we can provide one kind of flow and nutrients to make one side more bone-like, so that it will integrate with the underlying bone; and on the other side we can provide a different kind of flow stimulation and different biochemicals, and that will give us, we think, the kind of smooth, lubricating, low-friction surface that we rely on in all our joints.”

Secret Ingredients

“We and others are also working on engineering the scaffold that the tissue grows upon, which may also help address the polar issue,” Wick continues. “When I say scaffold, what I mean is a highly porous structure that acts as the skeleton for the tissue cells to grow on. For example, in our bioreactor for making blood vessels, the cells are placed on a polymer scaffolding that’s basically 90 percent porous. If you engineer it right, the cells are happy to stick to it.” Wick’s team begins growing a vessel wall by applying one set of cells and nutrients to the scaffolding; later in the culturing process they add endothelial cells that will create the inner surface.

He notes that not all scaffolds are polymers. “Some have fibers, so the scaffold looks like a loosely woven fabric, and others might have a structure that’s more like a spine, with lots of interconnected pores. And other people, such as physicist Yogesh Vohra here at UAB, are making scaffolds that have very distinct nanostructured architectures. If we engineer the scaffolding to be different at different points, perhaps we can graft different chemicals onto the scaffold so that one side can have one kind of growth factor and the other side can have others.”

Most scaffolds are designed to degrade as the tissues grow. That’s because as the tissues grow, they are creating their own natural scaffolding: a mysterious molecular medium called the extracellular matrix. What exactly exists in this area in between the cells of a tissue, and what all of its functions are, is still something of an enigma to scientists. But all agree that it is integral to any tissue type, as well as to successful tissue engineering.

“A lung cell, a heart cell, a bone cell—they’re all surrounded by molecules, and those molecules comprise the extracellular matrix,” says Joanne Murphy-Ullrich, Ph.D., the director of the Cell Adhesion and Matrix Research Center at UAB. “These are proteins and carbohydrate structures that determine a cell’s shape and also the mechanical properties of the cell, which regulate what genes are turned on within the cell. So this matrix—and also how the cell adheres to this matrix—really influences whether a stem cell becomes a bone cell or a cartilage cell.”

Murphy-Ullrich and other researchers in the center provide the molecular backbone for regenerative medicine work at UAB. “The matrix is also a repository for different growth factors that could stimulate cell division,” she says. “UAB has been historically strong in terms of people who study these kinds of proteins and matrix molecules—how they interact with cells, and how their interaction affects cell differentiation, cell growth, whether cells live or die.” These scientists can use their molecular “know-how” to help engineers develop the best conditions to allow the mysterious matrix glue to grow.

“Engineers are the ones who can design exciting biomaterials to use for regenerative strategies,” says Murphy-Ullrich. “We can warn them when a scaffolding that has the mechanical strength properties is lacking properties that will attract the stem cells to adhere to it and grow on it. So we really need a close working relationship and a good collaborative balance between our disciplines to get the process right.”

Cooking for the Masses

Once the best “specs” for growing a certain tissue type are identified, the bioreactor can be used to produce the tissues in mass quantities. “We’re in the process of learning the best scale-up,” Wick says. “We could have a factory with 2,000 bioreactors that each make one piece of tissue, or our factory could have one bioreactor that makes 2,000 pieces of tissue. The optimum is probably somewhere in between—we’ll use computer modeling to figure that out.”

The bioreactor also could be used to preserve tissues after they’re made, until they’re ready to be delivered to a surgeon for implantation in a patient. “In the bioreactor we’ll make the tissue, then freeze it there. Later we’ll harvest it and package it—in most instances surgeons will need only one at a time—and we’ll ship it to an orthopaedic surgeon. The next question after that,” he says, “is how it should be reconstituted in the operating suite so that it’s a living tissue again. We hope to develop some simple bioreactor technologies to do that, as well.

“We have to think in terms of what will be straightforward,” Wick says, “because there aren’t going to be tissue engineers working in the operating room. You want to send surgeons something with simple instructions, so that while the surgeon and the surgical team are worrying about the myriad issues that a surgeon and his team have to worry about, getting this piece of tissue in a living state to put in the patient isn’t one of them.”

Mix Until Well Combined

Questions of storage, reconstitution, and packaging will be solved in time; at this point Wick’s team is just beginning to test the effectiveness of engineered cartilage in mice and rats. He says that a group in Canada and a group in Japan have had some success with heart-valve leaflets—even a handful of human trials have shown some success in those models. Which tissue types come into the market first will depend on which technology progresses far enough to make the implant safe and effective, as well as where the demand is great enough to make it worth the investment of money, time, and surgical skill to test it in humans. Those interrelated variables make it hard to predict with certainty at this point, says Wick.

“There are a lot of models that look really successful right now,” he explains, “but there are more challenges to come. You might make a tissue that functions appropriately but activates the immune system, for example. There are also animal models right now in which cartilage implants work, and they stay in place for a year or so, but there’s no integration with the surrounding tissue—you can clearly see the boundary between the implant and the bone, almost like a cork in a wine bottle.

“And there will be different tolerances for shortcomings in different models,” he notes, “because different tissues are going to have different criticality. When a blood vessel fails, that’s a big problem. If a piece of cartilage fails, that’s something the patient can survive.

“So we still have a lot of issues to examine,” he says: how to engineer, produce, store, and implant a tissue that will heal well and integrate with the surrounding tissue, while not setting off an immune response, and that will perform all the duties of the original tissue dependably.

“Once we answer all those questions,” he says with a smile, “then we’re done.”